Combination therapy for treating cancer

ABSTRACT

The invention relates to a combination comprising an antineoplastic agent, e.g. an antimetabolite antineoplastic agent and a type 1 serotonin receptor (HTR 1)modulator, e.g. a HTR1 antagonist. In addition the invention relates to a pharmaceutical composition comprising a combination of the invention and a pharmaceutically acceptable excipient. The invention also relates to the combination and pharmaceutical composition according to the invention for use in medicine, particularly for use in the prevention and/or treatment of a hematological malignancy.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Appl. 16/478,121, with 371(c)date of Jul. 15, 2019, which issued as U.S. Pat. No. 11,446,321, on Sep.20, 2022, which is the U.S. national phase application of InternationalApplication No. PCT/EP2018/050876, filed Jan. 15, 2018, which claims thepriority of European Appl. No. 17382015.0, filed on Jan. 16, 2017, allof which are hereby incorporated by reference in their entireties.

TECHNICAL FIELD OF THE INVENTION

The invention is related to the field of cancer treatment and toaugmentation of anti-neoplastic drug efficacy.

BACKGROUND OF THE INVENTION

There are many antineoplastic agents available for treatment ofneoplastic diseases. The design or selection of a chemotherapeutic drugfor antineoplastic treatment must take into account many factors,depending on the tumor type, the physical condition of the patient andthe progression of tumor growth in the patient. Another factor toconsider in identifying an effective antineoplastic agent is the degreeof selectivity of the agent’s cytotoxic effect for the tumor cell overnormal host cells.

Toxicity is a major concern for anticancer drugs. These compoundspresent a narrow therapeutic index, with a small difference between thedoses. Approaches to the reduction of chemotherapy-induced toxicityinclude dose reduction, use of alternate drugs or their analogues,growth factors, and cytoprotective agents.

In addition to the pharmacokinetic changes that occur with age,pharmacodynamic changes such as altered intracellular metabolism of drugand decreased ability to repair DNA occur. With age, the toxicity ofnormal tissues is altered, and the risk and severity of myelodepression,mucositis, central and peripheral neurotoxicity, and cardiotoxicity alsoappear to increase.

Newly approved anticancer drugs are associated with increased toxicity,except for agents with a specific molecular target on cancer cells.Management of toxicity leads to a small increase in overall cost oftreatment and such toxicity can limit the net benefit of the treatments.Indeed, patients with a poor Eastern Cooperative Oncology Group (ECOG)performance status (ECOG> o = 3) do not receive intensive chemotherapytreatment as these patients do not tolerate chemotherapy based on thesevere side effects of this treatment. For instance, treatment withchemotherapy such as azacitidine, cytarabine or decitabine gives severeside effects like infections, blood and lymphatic disorders, nervoussystem disorders, respiratory disorders, gastrointestinal disorders andpyrexia among others. Unwanted side effects may lead to thediscontinuation of the optimal treatment regimen and the initiation of aless aggressive and less effective low-dose chemotherapeutic treatment,which is based on palliative care.

In an attempt to reduce the toxic effect of the antineoplastic agents,some synergistic combinations allowing the administration of lower dosesof the antineoplastic agent have been developed. The combination ofdoxorubicin, which produces cardiotoxicity, and dihydroartemisin (Wu GS.et al., Pharmacol Rep. 2013;65(2):453-9; US20160175331A1) showssynergistic effect so the same anticancer effect is obtained with lowdoses of the anticancer agent. In addition, cisplatin combined withOldenlandia diffusa induces synergistic anticancer effect inosteosarcoma MG-63 cell line in vitro (Pu Feife. et al., Onco TargetsTher. 2016 Jan 11;9:255-63) and the combination of cisplatin with crocinreduces the amount of cisplatin needed as well as its toxic side effect(Li X. et al., Toxicol Lett. 2013 Aug 29;221(3):197-204).

WO2009016488 discloses serotonin receptor 2A (HTR2A) antagonists incombination with a secondary treatment for the treatment of cancer, forexample breast tumors, gliomas, hormone-refractory prostate cancer andurologic tumors, particularly bladder tumors. This document provides along list of possible secondary treatments (see paragraphs[0013]-[0014], [0042]-[0049]) as well as a long list of suitable HTR2Aantagonists (see paragraphs [0039]) and possible combinations. In theexperimental work of WO2009016488, only one combination is tested,mianserin and tamoxifene, in mice xenografted with breast tumor cells.Mianserin is an HTR2A antagonist which is a tetracyclic antidepressant.WO2016007647 discloses nanoparticulated glutaminase inhibitors for thetreatment of pancreatic cancer and other glutamine addicted cancers.BPTES (bis-2[5-(phenylacetamido)-1,3,4-thiadiazol-2-yl]ethyl sulfide) isa compound used as antiproliferative but have problems of solubility andmetabolic stability. To be effective as glutaminase inhibitor, acompound has to be able to cross cell membranes and reach mitochondriawhere glutaminase enzyme resides. Thus, in the experimental work of thisdocument, the aim is to identify novel glutaminase inhibitor structures,by maintaining BPES lipophilic structure to ensure compatibility withnanoparticle formulation. The experimental work provides a long list ofnewly synthetized BPTES analogs as glutaminase inhibitors as well asoptimization of nanoparticule formulations including these BPTES.Further, Example 5 page 40 describes the combination of BPTESnanoparticles with non-encapsulated gemcitabine. Apomorphine is simplymentioned in this document as a possible glutaminase inhibitor, but nodata supports this statement. The skilled in the art by reading thisdocument can conclude that delivery of glutaminase inhibitors, includingapomorphine, have to be enhanced by means of encapsulation innanoparticles in order to reach the enzyme within the cell structure.Further, the skilled in the art cannot conclude that nanoencapsulatedapomorphine can work as serotonin receptor (HTR) antagonist, i.e. doingits effect in the cell membranes where the serotonin receptor islocated. Alternatively expressed, by reading this document the skilledperson has in practice no motivation to use apomorphine withoutencapsulation and acting as HTR antagonist. With respect to the use of ananoencapsulated glutamine inhibitors in combination withchemotherapeutics, the document does not disclose any data supportingthe effect of a combination, not even in pancreatic cancer - i.e. thecombination related statements may be seen as mere statements that arenot supported by any significant verifiable experimental data.

Chhavi Sharma et al. describes that gemcitabine and ginger extractinfusion may improve the efficiency of cervical cancer treatment (Chhaviet al. African Journal of Biotechnology, 2009 pp. 7087-7093). LuqmanHakin et al. discloses that ginger potentiates the anti cancer effect of5-FU (Luqman Hakin et al. Asian Pacific Journal of Cancer Prevention,vol. 15 no. 11, 15 2014 pp. 4651-4657). Gingerols and shogaols (derivedfrom ginger) are described as HTR3 antagonists. HTR3 is the onlyserotonin receptor that is not a GPCR, but an ion channel. It isstructurally and functionally distinct from HTR1 and HTR2. Ginger isadministered to help in reducing vomiting and nausea produced bychemotherapeutics.

Thus there is a need in the field of cancer chemotherapy to identifycompositions by which the toxic side effects can be reduced withoutcompromising the anticancer efficacy.

SUMMARY OF THE INVENTION

The present disclosure provides in a first aspect, therapeuticcombinations comprising at least one antineoplastic agent, e.g. anantimetabolite antineoplastic agent, and at least one type 1 serotoninreceptor (HTR1) modulator, e.g. at least one HTR1 antagonist. The term“combination of the invention” as used throughout the specificationrefers to these combinations.

In a second aspect, the present disclosure also provides pharmaceuticalcompositions comprising a combination of the invention and at least onepharmaceutically acceptable excipient. The term “pharmaceuticalcomposition of the invention” as used throughout the specificationrefers to these pharmaceutical compositions.

In a third aspect also provided are combinations of the invention orpharmaceutical compositions of the invention for use in medicine.

In a fourth aspect also provided are combinations of the invention orpharmaceutical compositions of the invention for use in the preventionand/or treatment of cancer.

In a fifth aspect, also provided are combinations of the invention orpharmaceutical compositions of the invention for use in the preventionand/or treatment of a hematological malignancy, e.g., lymphomas,leukemias and myelomas and particularly, acute myeloid leukaemia,myelodysplastic syndrome and myeloproliferative neoplasm.

The present disclosure also provides methods to treat, prevent, orameliorate the symptoms of hematological malignancies, e.g., lymphomas,leukemias and myelomas and particularly, acute myeloid leukaemia,myelodysplastic syndrome and myeloproliferative neoplasm.

Also provided as articles of manufacture and kits comprising thecombinations or pharmaceutical compositions of the invention disclosedherein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. Cytarabine displayed a synergistic anti-neoplasticeffect combined with HTR1 antagonists on AML cells. MonoMac-1 cells werecultured for 72 h in a complete RPMI medium in the presence of 2, 5, 10µM apomorphine (APO) or methiothepin (MET), and 250, 500, 1000 nMcytarabine (CYT). FIG. 1A. Cell viability was measured by flow cytometry(correct FSC-SSC profile, 7-AAD-, Hoechst33342^(low)). Normalized numberof live cells is graphed. Bars represent the mean value and error barsrepresent SEM. *** p<0.001; ****p<0.0001. NLC: Number of live cells(referred to control). FIG. 1B. Combination index (Cl) value of eachcombination.

FIGS. 2A and 2B. Azacitidine displayed a synergistic anti-neoplasticeffect combined with HTR1 antagonists on AML cells. MonoMac-1 cells werecultured for 72 h in a complete RPMI medium in the presence of 2, 5, 10µM apomorphine (APO) or methiothepin (MET), and 100, 200, 1000 nMazacitidine (AZA). FIG. 2A. Cell viability was measured by flowcytometry (correct FSC-SSC profile, 7-AAD-, Hoechst33342^(low)).Normalized number of live cells is graphed. Bars represent the meanvalue and error bars represent SEM. *** p<0.001; ****p<0.0001. NLC:Number of live cells (referred to control). FIG. 2B. Combination index(Cl) value of each combination.

FIGS. 3A and 3B. Decitabine displayed a synergistic anti-neoplasticeffect combined with HTR1 antagonists on AML cells. MonoMac-1 cells werecultured for 72 h in a complete RPMI medium in the presence of 2, 5, 10µM apomorphine (APO) or methiothepin (MET), and 20, 40, 100 nMdecitabine (DEC). NLC: Number of live cells (referred to control). FIG.3A. Cell viability was measured by flow cytometry (correct FSC-SSCprofile, 7-AAD⁻, Hoechst33342^(low)). Normalized number of live cells isgraphed. Bars represent the mean value and error bars represent SEM. ***p<0.001; ****p<0.0001. FIG. 3B. Combination index (Cl) value of eachcombination.

FIG. 4 . Apomorphine and cytarabine synergistically acted as anti-AMLagents in vivo. Bulsulfan-conditioned NSG adult mice were intravenouslyinjected with 1×10⁶ MonoMac-1 AML cells. One week after transplantation,mice were treated with 5 mg/kg apomorphine (APO) and/or 30 mg/kgcytarabine (CYT) daily for 5 days. Mice were culled and bones wereharvested. Human leukemia cells were detected based on the expression ofhCD45 on the surface membrane by flow cytometry. Normalized number ofhuman AML cells against control-treated mice is represented. Barscorrespond to mean values and error bars to SEM. ** p<0.01; ****p<0.0001. hAML: # human AML cells (referred to 15 control). CTR:Control.

FIG. 5 . Resistance to cytarabine did not affect the sensitivity to HTR1antagonists. Parental and Cytarabine-resistant (CYT-R) HL-60 and KG-1AML cells were cultured at a 3.5 x 10⁵ cells/mL in complete RPMI in96-well plates. Apomorphine or methiothepin were added at theconcentration indicated. At 72 h post-treatment, cells were analyzed byflow cytometry to measure cell viability (correct FSC-SSC profile,7-AAD⁻, Hoechst33342^(low)). Normalized number of live cells againstvehicle-treated control samples is represented. Error bars representSEM. LC: #live cells (referred to control). APO: logarithm Apomorphineconcentration. MET: logarithm Methiothepin concentration. HL-60: HL-60cell line. HL-60 CYT-R: Cytarabine-resistant HL-60 cell line. KG-1: KG-1cell line. KG-1 CYT-R: Cytarabine-resistant KG-1 cell line.

FIGS. 6A, 6B, and 6C. HTR1A and HTR1B expression correlated withsensitivity to cytarabine treatment. Parental and Ara-C-resistant HL-60and KG-1 cells were stained on cell surface with HTR1A and HTR1B. Theexpression level was measured by flow cytometry. FIG. 6A. MeanFluorescence Intensity (MFI) of HTR1A and HTR1B. White bars, parentalAML cell lines; black bars, Ara-C-resistant AML cell lines. Barsrepresent the mean value; error bars represent SEM. FIG. 6B.Representative HTR1A and HTR1B expression histogram plot of parental(grey) and Ara-C-resistant cells (black line). FIG. 6C. Correlationbetween HTR1A and HTR1B expression (MFI) and EC50 for cytarabine. *p<0.5; ** p<0.01. MFIc: Mean Fluorescence Intensity referred to control.MFI: Mean Fluorescence Intensity. HL-60: HL-60 cell line. HL-60 CYT-R:Cytarabine-resistant HL-60 cell line. KG-1: KG-1 cell line. KG-1 CYT-R:Cytarabine-resistant KG-1 cell line. CYT: logarithm Cytarabineconcentration.

FIG. 7 . Treatment with HTR1 antagonists decreased Sp1 gene expression.HL-60 AML cells were treated with 10 µM apomorphine (APO), 10 µMmethiothepin (MET) or 5 µM SB224289 (SB9) for 72 h. Total RNA wasisolated and cDNA was obtained afterwards. Semi-quantitative PCR wasperformed to detect gene expression. Sp1 gene expression is representedas 2^((-ΔΔCt)) using GAPDH as the endogenous control. Bars represent themean value of triplicates. Error bars correspond to SEM. * p<0.05; ****p<0.0001. ddCt: 2^((-ΔΔCt)).

FIGS. 8A and 8B. Cladribine displayed a synergistic anti-neoplasticeffect combined with HTR1 antagonists on AML cells. MonoMac-1 cells werecultured for 72 h in a complete RPMI medium in the presence of 5, 10 µMapomorphine (APO) or methiothepin (MET), and 10, 27, 55 nM cladribine(CLD). FIG. 8A. Combination index (Cl) value of CLD and APO combination.FIG. 8B. Cl value of CLD and MET combination.

FIGS. 9A and 9B. Fludarabine displayed a synergistic anti-neoplasticeffect combined with HTR1 antagonists on AML cells. MonoMac-1 cells werecultured for 72 h in a complete RPMI medium in the presence of 5, 10 µMapomorphine (APO) or methiothepin (MET), and 200, 500, 1000 nMfludarabine (FLD). FIG. 9A. Cl value of FLD and APO combination. FIG.9B. Cl value of FLD and MET combination.

FIG. 10 . Methotrexate displayed a synergistic anti-neoplastic effectcombined with apomorphine on AML cells. MonoMac-1 cells were culturedfor 72 h in a complete RPMI medium in the presence of 5, 10 µMapomorphine (APO), and 2.5, 6.25, 12.5 nM methotrexate (MTX). Cl valueof MTX and APO combination.

DETAILED DESCRIPTION OF THE INVENTION

The present specification discloses a synergistic anti-cancer effect ofseveral antineoplastic agents, e.g. antimetabolite antineoplastic agentsin combination with type 1 serotonin receptor (HTR1) modulators, e.g.HTR1 antagonists, when used in vitro (Examples 1-3) and in vivo (Example4). Said synergistic effect allows reducing the dose of theantimetabolite antineoplastic agent administered, thus reducing thetoxic effect produced in the patient. Unwanted side effects due toantineoplastic agents may lead to the discontinuation of the optimaltreatment regimen and the initiation of a less aggressive and lesseffective low-dose antineoplastic treatment. The use of a HTR1antagonist in combination with an antineoplastic agent such ascytarabine, decitabine or azacitidine therefore allows for atherapeutically effective dose of antineoplastic agents to beadministered at lower levels, thereby avoiding the undesirable sideseffects usually associated with higher doses of antineoplastic agents.

It has been determined that the combination of an HTR1 antagonist and anantimetabolite antineoplastic agent is unexpectedly effective forkilling cancer cells. As shown herein, serotonin receptor antagonistssuch as apomorphine, methiothepin or SB-224289 are cytotoxic to cancercells and particularly in acute myeloid leukemia (AML). Serotoninreceptor antagonists at concentrations toxic to cancer cells have alsobeen found to have a relatively limited effect on normal stem cells suchas hematopoietic stem cells and normal hematopoietic mature blood cells.Furthermore, as shown in Examples 1-4 and 8-10, the combination of theHTR1 antagonist apomorphine, methiothepine and SB-224289 and theantineoplastic agents cytarabine, decitabine, azacitidine, cladribine,fludarabine and methotrexate resulted in a synergistic effect and asignificant reduction in the number of AML cancer cells. The synergisticeffect between HTR1 antagonists and antimetabolite antineoplastic agentsis also shown in an in vivo xenotransplantation assay where humanAML-bearing mice are treated with apomorphine and/or cytarabine (Example4 FIG. 4 ). Furthermore, resistance to chemotherapeutic (cytarabine) didnot influence the sensitivity to HTR antagonists, Thus, the synergisticeffect observed might be due to the mechanism of action of HTR1antagonists that induce cytarabine sensitivity (Example 5, FIG. 5 ).Indeed, chemotherapeutic (cytarabine) resistance is accompanied by anoverexpression of HTR1 on the surface, suggesting that HTR1 expressionmay provide survival advantages (Example 6, FIGS. 6A, 6B, and 6C). Inaddition, the inventors have observed that HTR1 antagonists induce adecreased expression of SPI1 gene, a gene well-known for being relatedwith drug resistance whose downregulation produce antineoplastic agentsensitivity. Thus, HTR1 antagonists make cells more sensitive toantineoplastics (Example 7, FIG. 7 ).

I. Compositions Comprising an Antimetabolite Antineoplastic Agent and aType 1 Serotonin Receptor (HTR1) Antagonist

In some aspects, the present disclosure provides combinations comprisingat least one antineoplastic agent, e.g., an antimetaboliteantineoplastic agent and at least one HTR1 modulator, e.g., an HTR1antagonist. According to the invention the expression “combination”stands for the various combinations of compounds (A) and (B), forexample in a composition, in a combined mixture composed from separateformulations of the single active compounds, such as a “tank-mix”, andin a combined use of the single active ingredients when applied in asequential manner, i.e. one after the other with a reasonably shortperiod, such as a few hours or days or in simultaneous administration.In the present invention, compound (A) refers to a therapeuticallyeffective amount of at least one antineoplastic agent, e.g. anantimetabolite antineoplastic agent, or a pharmaceutically acceptablesalt thereof and compound (B) refers to at least one HTR1 modulator e.g.a HTR1 antagonist or a pharmaceutically acceptable salt thereof.Preferably the order of applying the compounds (A) and (B) is notessential for working the present invention.

A combination of at least one antineoplastic agent, e.g. anantimetabolite antineoplastic agent and at least one HTR1 modulator,e.g., an HTR1 antagonist can be formulated for its simultaneous,separate or sequential administration. Particularly, if theadministration is not simultaneous, the compounds are administered in aclose time proximity to each other. Furthermore, compounds areadministered in the same or different dosage form or by the same ordifferent administration route, e.g., one compound can be administeredtopically and the other compound can be administered orally. Suitably,both compounds are administered orally.

The combination of the two compounds, i.e. an antineoplastic agent,e.g., an antimetabolite antineoplastic agent and the HTR1 modulator,e.g., an HTR1 antagonist can be administered:

-   as a combination that is being part of the same medicament    formulation, the two compounds being then administered always    simultaneously.-   as a combination of two units, each with one of the substances    giving rise to the possibility of simultaneous, sequential or    separate administration.

In a particular embodiment, the antineoplastic agent, e.g. theantimetabolite antineoplastic agent is independently administered fromthe HTR1 modulator, e.g., the HTR1 antagonist (i.e., in two units) butat the same time.

In another particular embodiment, the HTR1 modulator, e.g. the HTR1antagonist is administered first, and then the antineoplastic agent e.g.the antimetabolite antineoplastic agent is separately or sequentiallyadministered.

In yet another particular embodiment, the antineoplastic agent, e.g. theantimetabolite antineoplastic agent is administered first, and then theHTR1 modulator, e.g. the HTR1 antagonist is administered, separately orsequentially, as defined.

“Antimetabolite antineoplastic agent”, as used herein, relates to acompound that interferes with the cancer cell’s metabolism. Some of themreplace essential metabolites without performing their functions, whileothers compete with essential components by mimicking their functionsand thereby inhibiting the manufacture of protein in the cell.Antimetabolites are cell cycle phase specific (S phase). Antimetaboliteantineoplastic agents and subgroups thereof are well-defined in theATC/DDD Index (established by the World Health 10 Organization), lastupdate 19.12.2016.

In a particular embodiment, the antimetabolite antineoplastic agent isselected from the group consisting of a folic acid analogue and anucleoside analogue. In a particular embodiment, the antimetaboliteantineoplastic agent is a folic acid analogue. “Folic acid analogue”, asused herein, is intended to encompass any analogue of folic acid thatcontains at least one carboxyl group and that acts as an antimetaboliteby interfering with normal folic acid dependent metabolic processes,thus producing the symptoms of a folic acid deficiency.

In a more particular embodiment, the folic acid analogue is selectedfrom the group consisting of methotrexate, raltitrexed, pemetrexed andpralatrexate. In a more particular embodiment, the folic acid analogueis methotrexate.

“Methotrexate”, as used herein, relates to the compound(2S)-2-[(4{[(2,4-Diaminopteridin-6-yl)methyl](methyl)amino}benzoyl)amino]pentanedioicacid having the CAS number 59-05-2.

In another particular embodiment, the antimetabolite antineoplasticagent is a nucleoside analogue.

“Nucleoside analogue”, as used herein, relates to a compound thatresembles naturally occurring nucleosides and acts by causingtermination of the nascent DNA chain.

In a particular embodiment, the nucleoside analogue is a purineanalogue.

“Purine analogue”, as used herein, relates to a compound that mimics thestructure of metabolic purines. In a particular embodiment, the purineanalogue is selected from the group consisting of mercaptopurine,tioguanine, cladribine, fludarabine, clofarabine and nelarabine. In amore particular embodiment, the purine analogue is mercaptopurine. Inanother particular embodiment the purine analogue is cladribine. Inanother particular embodiment, the purine analogue is fludarabine.

“Mercaptopurine”, as used herein, relates to the compound 3,7dihydropurine-6-thione having the CAS number 50-44-2.

“Cladribine”, as used herein, relates to the compound5-(6-Amino-2chloro-purin-9-yl)-2-(hydroxymethyl)oxolan-3-ol having theCAS number 429163-8.

“Fludarabine”, as used herein, relates to the compound[(2R,3R,4S,5R)-5-(6-amino-2-fluoro-purin-9-yl)-3,4-dihydroxy-oxolan-2-yl]methoxyphosphonicacid having the CAS number 75607-67-9.

In another particular embodiment, the nucleoside analogue is a“pyrimidine analogue”.

“Pyrimidine analogue”, as used herein, relates to a compound whichmimics the structure of metabolic pyrimidines. Pyrimidine analogues arewell-defined in the ATC/DDD Index or Classification System Anatomical,Therapeutic, Chemical Code (established by the World Health Organizationand adopted in Europe) group. A pyrimidine analogue can be a thymidineanalogue or a cytidine analogue. “Thymidine analogue”, as used herein,relates to a compound which mimics the structure of thymidine, anucleoside molecule formed when thymine is attached to deoxyribose via aβ-N3-glycosidic bond. “Cytidine analogue”, as used herein, relates to acompound which mimics the structure of cytidine, a nucleoside moleculeformed when cytosine is attached to a ribose ring via a β-N1-glycosidicbond.

In a particular embodiment, the pyrimidine analogue is selected from thegroup consisting of cytarabine, fluorouracil, tegafur, carmofur,gemcitabine, capecitabine, azacitidine, decitabine and trifluridine.

In a particular embodiment, the pyrimidine analogue is cytarabine. Inanother particular embodiment, the pyrimidine analogue is azacitidine.In another particular embodiment, the pyrimidine analogue is decitabine.

“Cytarabine” or cytosine arabinoside or ara-C (Cytosar-U or Depocyt)refers to the compound 4-amino-1-[(2R,3S,4R,5R)-3,4-dihydroxy-5(hydroxymethyl)oxolan-2-yl] pyrimidin-2-one having the CAS-number147-94-4.

“Azacitidine” refers to the compound4-Amino-1-β-D-ribofuranosyl-1,3,5triazin-2(1H)-one having the CAS number320-67-2.

“Decitabine” or 5-aza-2′-deoxycytidine refers to the compound4-Amino-1-(2-deoxy-β-D-erythro-pentofuranosyl)-1,3,5-triazin-2(1H)-onehaving the CAS number 2353-33-5.

The combination of the invention, in addition to the antimetaboliteantineoplastic agent, comprises a HTR1 antagonist.

The combination of the invention can comprise more than oneantimetabolite antineoplastic agent and more than one HTR1 antagonist.

“Serotonin receptors”, also known as HTR, 5-hydroxytryptamine receptors,5-HT receptors or 5-HTR, as used herein, are a group of Gprotein-coupled receptors (GPCRs) and ligand-gated ion channels (LGICs)mostly found in the central and peripheral nervous systems.

“Serotonin receptor (HTR) antagonist” refers to a compound that binds tothe 5-HT receptor and lacks any substantial ability to activate thereceptor itself. An antagonist can thereby prevent or reduce thefunctional activation or occupation of the receptor by an agonist or thenatural ligand when the agonist is present. The term “antagonist of the5-HT receptor”, as used herein, is intended to encompass both neutralantagonists and inverse agonists. A “neutral antagonist” is a compoundthat blocks the action of the agonist but has no effect on intrinsic orspontaneous receptor activity. An “inverse agonist” is able to bothblock the action of the agonist at the receptor and attenuates theconstitutive activity of the receptor. The term “antagonist” alsoincludes competitive antagonists, which are drugs that bind to the samesite as the natural ligand; noncompetitive antagonists which bind to adifferent site on the receptor than the natural ligand; reversibleantagonists which bind and unbind the receptor at rates determined byreceptor-ligand kinetics; and irreversible antagonists which bindpermanently to the receptor either by forming a covalent bond to theactive site or just by binding so tightly that the rate of dissociationis effectively zero.

The term “HTR1” or “type 1 HTR” or “type 1 5-HT receptor” or “type 15HTR” or “5-HT1 receptor” or “5-HTR1”, as used herein, relates to asubfamily of 5-HT receptors that bind the endogenous neurotransmitterserotonin (5-hydroxytryptamine, 5-HT). The 5-HT1 receptor subfamilyconsists of five G protein-coupled receptors (GPCRs) that are coupled toGi/Go and the term includes HTR1A, HTR1B, HTR1D, HTR1E, and HTR1F. Thesereceptors mediate inhibitory neurotransmission by decreasing cellularlevels of cAMP. The complete protein sequence for human type 1A 5-HTreceptor has the UniProt accession number P08908 (Nov. 30, 2016). Thecomplete protein sequence for human type 1B 5-HT receptor has theUniProt accession number P28222 (Nov. 30, 2016). The complete proteinsequence for human type 1D 5-HT receptor has the UniProt accessionnumber P28221 (Nov. 2, 2016). The complete protein sequence for humantype 1E 5-HT receptor has the UniProt accession number P28566 (Nov. 2,2016). The complete protein sequence for human type 1F 5-HT receptor hasthe UniProt accession number P30939 (Nov. 2, 2016).

In a particular embodiment, the HTR1 antagonist is type 1A HTRantagonist. In another particular embodiment, the HTR1 antagonist istype 1B HTR antagonist.

The person skilled in the art knows how to determine the affinity of aparticular molecule for a type 1 HTR and also to determine if thisparticular molecule is an antagonist of said receptor. Particularsuitable assays are radioligand binding assays to determine the bindingaffinity, and functional studies of the mobilization of secondmessengers. For example, the HTR affinity of a molecule can bedetermined using the methodology described by Millan et al. (Millan etal. J Pharmacol Exp Ther. 2002;303(2):791-804) (radioligand bindingassay) An assay to assess if a compound is a type 1 HTR antagonist isthe determination of the Gi activation status and measuring the cAMPproduction and activation of adenylyl cyclase (Nichols D.E. and NicholsC. E. Chem Rev, 2008;108(5): 1614-41). The activity of type 1 HTR can bedetermined by detecting decreasing levels of cAMP (Williams C. Nat RevDrug Discovery, 2004;3(2):125-35) and increasing levels of phosphor-Akt(Suni MA. and Maino VC. Methods Mol Biol 2011;717:155-69).

Through the examples, different compounds are provided for complyingwith this ability to antagonize HTR1. Further to said compounds, bymeans of the methods described before, it is plausible to identify othercompounds with the same ability to antagonize HTR1. It is alsodemonstrated by means of the Examples, that this ability of antagonizingHTR1 in combination with the administration of an antineoplastic agent,e.g. an antimetabolite antineoplastic agent is therapeuticallybeneficial according to the purposes of the invention. Thus, althoughsome specific compounds have been tested and identified with thisbeneficial effect, there is no reason to limit the scope of theinvention to such compounds because all the steps of the method to getother good candidates are plausibly described herein.

In a more particular embodiment, the type 1A HTR antagonist is selectedfrom the group consisting of GR 125,743; GR 218,231; MPDT; NAN 190;pizotifen; p-MPPI; Rec 15/3079; repinotan; robalzotan; SDZ-216525;tertatolol; UH301; WAY-100135; apomorphine; cyamemazine; flurocarazolol;metergoline; methiothepin; methysergide; SB 224289 and tiospirone. In amore particular embodiment, the type 1A HTR antagonist is a 1A HTRsubtype-specific antagonist, more particularly is a 1A HTRsubtype-specific antagonist selected from the group consisting of GR125,743; GR 218,231; MPDT; NAN 190; pizotifen; p-MPPI; Rec 15/3079;repinotan; robalzotan; SDZ-216525; tertatolol; UH301 and WAY-100135.

In another particular embodiment the type 1B HTR antagonist is selectedfrom the group consisting of GR-55562; ocaperidone; SB 272183; SB649915; SB 714786; SB 236057; apomorphine; cyamemazine; flurocarazolol;metergoline; methiothepin; methysergide; SB 224289 and tiospirone. In amore particular embodiment, the type 1B HTR antagonist is a 1B HTRsubtype-specific antagonist, more particularly is a 1B HTRsubtype-specific antagonist selected from the group consisting ofGR-55562; ocaperidone; SB 272183; SB 649915; SB 714786 and SB 236057.

In a particular embodiment, the combination of the invention comprisesat least one type 1 HTR antagonist. In a more particular embodiment, thecombination of the invention comprises apomorphine. In anotherparticular embodiment, the combination of the invention comprisesmethiothepin. In another embodiment, the combination of the inventioncomprises amperozide. In another embodiment, the combination of theinvention comprises mianserin.

“Apomorphine”, as used herein, refers to the compound(6aR)-6-methyl-5,6,6a,7-tetrahydro-4H-dibenzo[de,g]quinoline-10,11-diol,CAS number 314-19-2.

“Methiothepin” or metitepine, refers to the compound1-methyl-4-(8methylsulfanyl-5,6-dihydrobenzo[b][1]benzothiepin-6-yl)piperazine,CAS number 74611-28-2.

In a particular embodiment, the combination comprises a pyrimidineanalogue and a type 1A HTR antagonist. In another embodiment, thecombination of the invention comprises a pyrimidine analogue and a type1A HTR antagonist. In another embodiment, the combination of theinvention comprises a pyrimidine analogue and a type 1B HTR antagonist.

In an embodiment, the combination of the invention comprises apyrimidine analogue and apomorphine. In an embodiment, the combinationof the invention comprises a pyrimidine analogue and methiothepin.

In particular embodiments, the combination of the invention comprisescytarabine and apomorphine; cytarabine and methiothepin; azacitidine andapomorphine; azacitidine and methiothepin; decitabine and apomorphine;or decitabine and methiothepin.

In another particular embodiment, the combination of the inventioncomprises a folic acid analogue and a type 1A HTR antagonist. In anotherparticular embodiment, the combination of the invention comprises afolic acid analogue and a type 1B HTR antagonist. In another particularembodiment, the combination of the invention comprises a folic acidanalogue and apomorphine.

In another particular embodiment, the combination of the inventioncomprises a folic acid analogue and methiothepin.

In another particular embodiment, the combination of the inventioncomprises a purine analogue and a type 1A HTR antagonist. In anotherparticular embodiment, the combination of the invention comprises apurine analogue and a type 1B HTR antagonist. In another particularembodiment, the combination of the invention comprises a purine analogueand apomorphine. In another particular embodiment, the combination ofthe invention comprises a purine analogue and methiothepin.

In another particular embodiment, the combination of the inventioncomprises methotrexate and apomorphine, methotrexate and methiothepin,mercaptopurine and apomorphine, mercaptopurine and methiothepin,cladribine and apomorphine, cladribine and methiothepin, fludarabine andapomorphine or fludarabine and methiothepin.

In the present invention, when referred to a particular compound is alsointended to encompass the pharmaceutically acceptable salt of saidcompound.

The term “pharmaceutically acceptable salt thereof”, as used herein,refers to derivatives of the compounds of the invention wherein theparent compound is modified by making acid or base salts thereof.Examples of pharmaceutically acceptable salts include, but are notlimited to, mineral or organic acid salts of basic residues such asamines; alkali or organic salts of acidic residues such as carboxylicacids; and the like. The pharmaceutically acceptable salts include theconventional non-toxic salts or the quaternary ammonium salts of theparent compound formed, e.g., from non-toxic inorganic or organic acids.For example, such conventional non-toxic salts include, but are notlimited to, those derived from inorganic and organic acids selected from1,2-ethanedisulfonic, 2-acetoxybenzoic, 2-hydroxyethanesulfonic, acetic,ascorbic, benzenesulfonic, benzoic, bicarbonic, carbonic, citric,edetic, ethane disulfonic, ethane sulfonic, fumaric, glucoheptonic,gluconic, glutamic, glycolic, glycollyarsanilic, hexylresorcinic,hydrabamic, hydrobromic, hydrochloric, hydroiodide, hydroxymaleic,hydroxynaphthoic, isethionic, lactic, lactobionic, lauryl sulfonic,maleic, malic, mandelic, methanesulfonic, napsylic, nitric, oxalic,pamoic, pantothenic, phenylacetic, phosphoric, polygalacturonic,propionic, salicylic, stearic, subacetic, succinic, sulfamic,sulfanilic, sulfuric, tannic, tartaric, and toluenesulfonic.

The pharmaceutically acceptable salts of the compounds of the inventioncan be synthesized from the parent compound that contains a basic oracidic moiety by conventional chemical methods. Generally, such saltscan be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two;generally, non-aqueous media like ether, ethyl acetate, ethanol,isopropanol, or acetonitrile are useful. Lists of suitable salts arefound in Remington’s Pharmaceutical Sciences, 18th ed., Mack PublishingCompany, Easton, Pa., 1990, p. 1445.

II. Compositions Comprising at Least an Antineoplastic Agent and atLeast a HTR1 Modulator

In some aspects the present disclosure provides compositions comprisingat least an antineoplastic agent, e.g., an antimetabolite antineoplasticagent, and at least one HTR1 modulator, e.g., an HTR1 antagonist (suchas a small molecule antagonist, an antibody or antigen binding portionthereof capable of specifically binding to HTR1, a polypeptideantagonist, a nucleic acid such as an siRNA, or any HTR1 antagonistsknown in the art).

II.i. HTR1 Modulators

As said before, combinations are provided comprising at least oneantineoplastic agent, e.g. an antimetabolite antineoplastic agent, andat least one HTR1 modulator, e.g. at least one HTR1 antagonist. The term“modulator”, as used herein, refers to a compound modulating theactivity of the HTR1 (e.g. by binding to the HTR1 and lacking anysubstantial ability to activate the receptor itself; or by preventing orreducing the expression of HTR mRNA or HTR protein). The term modulatorincludes, without limitation, selective inhibitors for HTR1 or for atype of HTR1, non-selective inhibitors that are also capable of actingas inhibitors on other HTR types or on other receptors (e.g. dopaminereceptor), antagonists of HTR1, antibodies against HTR1, compounds whichprevent expression of HTR1 and compounds which lead to reduced mRNA orprotein levels of HTR1. In a preferred embodiment the modulator is anantagonist. The HTR1 modulator can be, among others, a protein, apeptide, interference RNA, an antisense oligonucleotide or a smallorganic molecule. In a particular embodiment, the HTR1 modulator isHTR1A modulator. In another particular embodiment, the HTR1 modulator isHTR1B modulator.

In other particular embodiments, the HTR1 antagonist is a compound thatis also antagonist of dopamine receptor, selected from the groupconsisting of: Mesoridazine, Sertindole, Butaclamol, Raclopride,Pimozide, S33084 and SB277011-A.

In an embodiment, the HTR1 modulator is an inhibitory antibody. The term“inhibitory antibody” is understood to mean, according to the presentinvention, an antibody that is capable of binding to HTR1 provoking theinhibition of the activation of said receptor by its natural ligand.Antibodies can be prepared using any method known by a person skilled inthe art. Thus, polyclonal antibodies are prepared by immunization of ananimal with the protein aimed to be inhibited. Monoclonal antibodies canbe prepared using the method described by Kohler, Milstein et al(Nature, 1975, 256: 495). Those antibodies capable of inhibiting HTR1activity using the abovementioned assays for determination of HTR1activity will be selected. Suitable antibodies in the present inventioninclude intact antibodies which comprise an antigen-binding variableregion and a constant region, fragments “Fab”, “F(ab′)2”, “Fab′”, Fv,scFv, diabodies and bispecific antibodies.

In another embodiment, the modulator is an interference RNA. As usedherein, the term “interference RNA” or “iRNA” refers to RNA moleculescapable of silencing the expression of HTR1 gene or of any gene neededfor HTR1 function. To that end, iRNA are typically double-strandedoligonucleotides having at least 30 base pairs in length, and they morepreferably comprise about 25, 24, 23, 22, 21, 20, 19, 18 or 17ribonucleic acid base pairs. Several different types of molecules havebeen used effectively in iRNA technology including small interfering RNA(siRNA) sometimes known as short interference RNA or silencer RNA, microRNA (miRNA) which normally differ from siRNA because they are processedfrom single-stranded RNA precursors and they are shown to be onlypartially complementary to the target mRNA and short hairpin RNA(shRNA).

Small interfering RNA (siRNA) agents are capable of inhibiting targetgene expression by interfering RNA. siRNAs can be chemicallysynthesized, or can be obtained by in vitro transcription, o can besynthesized in vivo in target cell. Typically, siRNAs consist of adouble-stranded RNA from 15 to 40 nucleotides in length and can containa protuberant region 3′ and/or 5′ from 1 to 6 nucleotides in length.Length of protuberant region is independent from total length of siRNAmolecule. siRNAs act by post-transcriptional degradation or silencing oftarget messenger.

siRNA can be denominated shRNA (short hairpin RNA) characterized in thatthe antiparallel strands that form siRNA are connected by a loop orhairpin region. siRNAs are constituted by a short antisense sequence (19to 25 nucleotides) followed by a loop of 5-9 nucleotides, and the sensestrand. shRNAs can be encoded by plasmids or virus, particularlyretrovirus and, more particularly, retrovirus and under the control ofpromoters such as U6 promoter for RNA polymerase III.

The siRNAs of the invention are substantially homologous to type 1 HTRmRNA or its protein-coding genome sequence. The term “substantiallyhomologous” is understood to mean that siRNAs have a sequencesufficiently complementary or similar to target mRNA so that siRNA canbe able to provoke mRNA degradation by RNA interference. Suitable siRNAsto provoke interference include siRNAs formed by RNA, as well as siRNAscontaining chemically different modifications such as:

-   -siRNAs in which the links between nucleotides are different from    those appearing in nature, such as phosphorothioate links.-   -Stranded-RNA conjugates with a functional reagent, such as a    fluorophore.-   -Modification of the ends of RNA strands, particularly the 3′ end by    the combination with different functional hydroxyl groups at    2′-position.-   -Sugar-modified nucleotides such as O-alkylated radicals at    2′-position such as 2′-O-methylribose or 2′-O-fluororibose.-   -Base-modified nucleotides such as halogenated bases (e.g.,    5-bromouracil and 5-iodouracil) or alkylated bases (e.g.,    7-methyl-guanosine).

The siRNAs and shRNAs of the invention can be obtained using a series oftechniques known to a person skilled in the art. For example, siRNA canbe chemically synthesized from protected ribonucleoside phosphoramiditesin a conventional DNA/RNA synthesizer. Alternatively, siRNA can beproduced by recombinant dicer from plasmid and viral vectors, where thecoding region of siRNA strand or strands is under operative control ofRNA polymerase III promoters. RNase Dicer processes shRNA into siRNA incells.

The region which is taken as a basis for the design of siRNA is notlimitative and can contain a region of coding sequence (between theinitiation codon and the termination codon) or, alternatively, cancontain sequences from the 5′ or 3′ untranslated region, preferably from25 to 50 nucleotides in length and in any position in 3′ position withregard to the initiation codon. A procedure for siRNA design involvesthe identification of sequence motive AA(N19)TT wherein N can be anynucleotide in the sequence of interest and the selection of those thatexhibit a high content in G/C. If said sequence motive is not found, itis possible to identify sequence motive NA(N21) wherein N can be anynucleotide.

In another embodiment, the HTR1 modulator is an antisenseoligonucleotide specific to HTR, i.e., molecules whose sequence iscomplementary to mRNA coding for HTR1, i.e., complementary to cDNAcoding strand. The antisense oligonucleotide can be complementary to acomplete coding region or a region of same including both the codingregion and the 5′ and 3′ untranslated regions. The antisenseoligonucleotides can consist of 5, 10, 15, 20, 25, 30, 35, 40, 45, 50 ormore nucleotides in length. The antisense oligonucleotides can beobtained by chemical synthesis or by enzymatic binding reactions widelyknown to a person skilled in the art. For example, an antisenseoligonucleotide can further contain modified nucleotides which increaseits biological stability or the stability of the bicatenary DNA-RNAcomplexes formed between the antisense oligonucleotide and the targetpolynucleotide, such as phosphorothioate derivatives, peptide nucleicacids and acridine-substituted oligonucleotides. Modifiedoligonucleotides that can be used for the preparation of antisensenucleic acids include 5-fluorouracil, 5-bromouracil, 5-chlorouracil,5-iodouracil, hypoxanthine, xanthine, 4-acetyl-cytosine,5-(carboxyhydroxylmethyl) uracil,5-carboxymethylaminomethyl-2-thiouridine, 5-carboxymethyl-aminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine,N6-isopentenyladenine, 1-methylguanine, 1-methylinosine,2,2dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine,5-methylcytosine, N6-adenine, 7-methylguanine,5-methylaminomethyluracil, 5methoxyaminomethyl-2-thiouracil,beta-D-mannosylqueosine, 5'methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid,pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil,2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethyl ester, 3-(3-amino-3-N-2-carboxypropyl)uracil, and2,6-diaminopurine. Alternatively, the antisense nucleic acid can beproduced biologically using an expression vector in which theantisense-oriented nucleic acid has been cloned.

Another group of modulators that can be used in the present inventionare catalytically active nucleic acids known as ribozymes. Ribozymescomprise a catalytic region and a second region whose sequence iscomplementary to target nucleic acid and confers substrate specificityon the ribozyme. After the interaction between the ribozyme and itssubstrate by hybridization and coupling between complementary regions oftarget nucleic acid and ribozyme, an activation of the catalytic regionis produced provoking the inter- or intramolecular rupture of targetnucleic acid. Basic considerations for the design of ribozymes arewidely known to a person skilled in the art (see, e.g., Doherty andDoudna (Annu. Rev. Biophys. Biomol. Struct. 2001; 30:457-75).

Other compounds capable of modulating HTR1 and/or the expression thereofthat can be used in the invention include aptamers and spiegelmers.

Aptamers and spiegelmers are single-stranded or double-stranded D- orL-nucleic acids that specifically bind to the protein resulting in amodification of the biological activity of the protein. Aptamers andspiegelmers are 15 to 80 nucleotides in length and, preferably, 20 to 50nucleotides in length.

Suitable methods for determining whether a modulator is capable ofdecreasing mRNA levels include, without limitation, standard assays fordetermining mRNA expression levels such as qPCR, RT-PCR, RNA protectionanalysis, Northern blot, RNA dot blot, in situ hybridization, microarraytechnology, tag based methods such as serial analysis of gene expression(SAGE) including variants such as LongSAGE and SuperSAGE, microarrays,fluorescence in situ hybridization (FISH), including variants such asFlow-FISH, qFiSH and double fusion FISH (D-FISH), and the like.

Suitable methods for determining whether a modulator acts by decreasingthe HTR1 protein levels include the quantification by means ofconventional methods, e.g., using antibodies with a capacity tospecifically bind to the proteins encoded by the gene (or to fragmentsthereof containing antigenic determinants) and subsequent quantificationof the resulting antibody-antigen complexes.

An HTR1 modulator according to the invention may inhibit HTR1 activityby at least 5%, at least 10%, at least 25%, at least 50%, at least 75%,or at least 90%, and all ranges between 5% and 100%. Suitable methodsfor determining whether a modulator acts by decreasing HTR1 activityhave been previously described.

In particular embodiments, the HTR1 modulator is selected from the groupconsisting of the compounds of formula I disclosed in EP 0687472 A2,inhibitory antibodies of HTR, an interference RNA specific for the type1 HTR sequences, an antisense oligonucleotide specific for the type 1HTR sequences, a ribozyme or DNA enzyme specific for the type 1 HTRsequences.

II.ii. Antineoplastic Agents

According to the ATC/DDD Index (update 19/12/2016), antineoplasticagents are codified as L01 therapeutic group. This group comprisespreparations used in the treatment of malignant neoplastic diseases. Inparticular embodiments the antineoplastic agent is a compound selectedfrom the compounds included in the subgroups of antineoplastic agentsgroup L01. Subgroups include L01A Alkylating agents, L01BAntimetabolites, L01C Plant alkaloids and other natural products, L01DCytotoxic antibiotics and related substances, and L01X Otherantineoplastic agents. Alkylating agents are classified in Nitrogenmustard analogues (e.g. cyclophosphamide, chlorambucil, melphalan,chlormethine, ifosfamide, trofosfamide, prednimustine, bendamustine),Alkyl sulfonates (e.g. busulfan, treosulfan, mannosulfan), Ethyleneimines (e.g. thiotepa, triaziquone, carboquone), Nitrosoureas, (e.g.carmustine, lomustine, semustine, streptozocin, fotemustine, nimustine,ranimustine) Epoxides (e.g. etoglucid) and Other alkylating agents (e.g.mitobronitol, pipobroman, temozolomide, dacarbazine). Plant alkaloidsand other natural products include Vinca alkaloids and analogues(vinblastine, vincristine, vindesine, vinorelbine, vinflunine,vintafolide), Podophyllotoxin derivatives (e.g. etoposide, teniposide),Taxanes (e.g. paclitaxel, docetaxel, paclitaxel poliglumex,cabazitaxel), and Other plant alkaloids and natural products (e.g.trabectedin). Cytotoxic antibiotics and related substances includeActinomycines (e.g. dactinomycin), Anthracyclines (e.g. doxorubicin,daunorubicin, epirubicin, aclarubicin, zorubicin, idarubicin,mitoxantrone, pirarubicin, valrubicin, amrubicin, pixantrone and relatedsubstances), and Other cytotoxic antibiotics (e.g. bleomycin,plicamycin, mitomycin, ixabepilone). Other antineoplastic agents includePlatinum compounds (e.g. cisplatin, carboplatin, oxaliplatin,satraplatin, polyplatillen), Methylhydrazines (e.g. procarbazine),Monoclonal antibodies for the treatment of cancer (e.g. edrecolomab,rituximab, trastuzumab, gemtuzumab, cetuximab, bevacizumab, panitumumab,catumaxomab, ofatumumab, ipilimumab, brentuximab, vedotin, pertuzumab,trastuzumab emtansine, obinutuzumab dinutuximab nivolumab pembrolizumabblinatumomab ramucirumab, necitumumab, elotuzumab, daratumumab,mogamulizumab, inotuzumab ozogamicin), Anecortave, indicated for thetreatment of exudative age-related macular degeneration, Sensitizersused in photodynamic/radiation therapy (e.g. porfimer sodium, methylaminolevulinic acid, temoporfin, efaproxiral), Protein kinase inhibitors(e.g. imatinib, gefitinib, erlotinib, sunitinib, sorafenib, dasatinib,lapatinib, nilotinib, temsirolimus, everolimus, pazopanib, vandetanib,afatinib, bosutinib, vemurafenib, crizotinib, axitinib, ruxolitinib,ridaforolimus, regorafenib, masitinib, dabrafenib, ponatinib,trametinib, cabozantinib, ibrutinib, ceritinib, lenvatinib, nintedanib,cediranib, palbociclib, tivozanib, osimertinib, alectinib, rociletinib,cobimetinib, midostaurin, olmutinib), and Other antineoplastic agents(e.g. amsacrine, asparaginase, altretamine, hydroxycarbamide,lonidamine, pentostatin, miltefosine, masoprocol, estramustine,tretinoin, mitoguazone, topotecan, tiazofurine, irinotecan,alitretinoin, mitotane, pegaspargase, bexarotene, arsenic trioxide,denileukin diftitox, bortezomib, celecoxib, anagrelide, oblimersen,sitimagene ceradenovec, vorinostat, romidepsin, omacetaxinemepesuccinate, eribulin, panobinostat, vismodegib, aflibercept,carfilzomib, olaparib, idelalisib, sonidegib, belinostat, ixazomib,talimogene laherparepvec, enetoclax, vosaroxin).

III. Pharmaceutical Compositions

In another aspect, the invention relates to a pharmaceutical compositioncomprising a combination according to the invention and apharmaceutically acceptable excipient.

The term “pharmaceutical composition” as used herein refers to apreparation which is in such form as to permit the biological activityof the active ingredients (i.e. the HTR1 modulator, e.g. the HTR1antagonist and the antineoplastic agent, e.g. an antimetaboliteantineoplastic agent) to be effective, and physiologically tolerable,that is, which contains no additional components which are unacceptablytoxic to a subject to which the composition would be administered. Suchcomposition can be sterile. Particularly, the term “pharmaceuticallyacceptable” means it is approved by a regulatory agency of a state orfederal government or is included in the U.S. Pharmacopoeia or othergenerally recognized pharmacopoeia for use in animals, and moreparticularly in humans.

Appropriate amounts of a compound of the combination of the invention asdefined above can be formulated with pharmaceutically acceptableexcipients and/or carriers to obtain a pharmaceutical composition foruse in medicine, particularly in preventing and/or treating cancer, moreparticularly in preventing and/or treating a hematological malignancy.

The term “excipient” refers to a vehicle, diluent or adjuvant that isadministered with the active ingredient. Such pharmaceutical excipientscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and similars. Water or salineaqueous solutions and aqueous dextrose and glycerol solutions,particularly for injectable solutions, are particularly used asvehicles. Suitable pharmaceutical vehicles are described in “Remington’sPharmaceutical Sciences” by E.W. Martin, 21st Edition, 2005; or“Handbook of Pharmaceutical Excipients”, Rowe C. R.; Paul J. S.; MarianE. Q., sixth Edition. Suitable pharmaceutically acceptable vehiclesinclude, e.g., water, salt solutions, alcohol, vegetable oils,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, surfactants, silicic acid, viscous paraffin, perfume oil,monoglycerides and diglycerides of fatty acids, fatty acid esterspetroetrals, hydroxymethyl cellulose, polyvinylpyrrolidone and similars.

The pharmaceutical compositions containing the combination of theinvention as defined above can occur at any pharmaceutical form ofadministration (e.g. in the form of, lyophilized powders, slurries,aqueous solutions, lotions, or suspensions) considered appropriate forthe selected administration route, including, but not limited to,systemic (e.g. intravenous, subcutaneous, intramuscular injection)intradermal, intraperitoneal, intranasal, epidural, topical, and oralroutes, for which it will include the pharmaceutically acceptableexcipients necessary for formulation of the desired method ofadministration. Additionally, it is also possible to administer thecomposition of the invention as defined above intranasally orsublingually which allows systemic administration by a non-aggressivemode of administration. Also, intraventricular administration can beadequate. A particular route of delivery is oral.

Those skilled in the art are familiar with the principles and proceduresdiscussed.

In a particular embodiment, the composition of the invention isadministered intravenously, and more particularly by infusion or bolusinjection. Where necessary, the combination of the invention iscomprised in a composition also including a solubilizing agent and alocal anesthetic to ameliorate any pain at the site of the injection.Generally, the ingredients are supplied either separately or mixedtogether in unit dosage form, e.g., as a dry lyophilized powder or waterfree concentrate in a hermetically sealed container such as an ampule orsachette indicating the quantity of active agent. Where the compositionis to be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water or saline. Wherethe composition is administered by injection, an ampule of sterile waterfor injection or saline can be provided so that the ingredients can bemixed prior to administration.

In cases other than intravenous administration, the composition cancontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. The composition can be a liquid solution, suspension, emulsion,gel, polymer, or sustained release formulation. The composition can beformulated with traditional binders and carriers, as would be known inthe art. Formulations can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharide, cellulose, magnesium carbonate, etc., inert carriershaving well established functionality in the manufacture ofpharmaceuticals. Various delivery systems are known and can be used toadminister a combination or pharmaceutical composition of the presentinvention including encapsulation in liposomes, microparticles,microcapsules and the like.

Solid dosage forms for oral administration can include conventionalcapsules, sustained release capsules, conventional tablets,sustained-release tablets, chewable tablets, sublingual tablets,effervescent tablets, pills, suspensions, powders, granules and gels. Atthese solid dosage forms, the active compounds can be mixed with atleast one inert excipient such as sucrose, lactose or starch. Suchdosage forms can also comprise, as in normal practice, additionalsubstances other than inert diluents, e.g. lubricating agents such asmagnesium stearate. In the case of capsules, tablets, effervescenttablets and pills, the dosage forms can also comprise buffering agents.Tablets and pills can be prepared with enteric coatings.

Liquid dosage forms for oral administration can include emulsions,solutions, suspensions, syrups and elixirs pharmaceutically acceptablecontaining inert diluents commonly used in the technique, such as water.Those compositions can also comprise adjuvants such as wetting agents,emulsifying and suspending agents, and sweetening agents, flavoring andperfuming agents.

Injectable preparations, e.g., aqueous or oleaginous suspensions,sterile injectable can be formulated according with the technique knownusing suitable dispersing agents, wetting agents and/or suspendingagents. Among the acceptable vehicles and solvents that can be used arewater, Ringer’s solution and isotonic sodium chloride solution. Sterileoils are also conventionally used as solvents or suspending media.

For topical administration combinations of the invention can beformulated as creams, gels, lotions, liquids, pomades, spray solutions,dispersions, solid bars, emulsions, microemulsions and similars whichcan be formulated according to conventional methods that use suitableexcipients, such as, e.g., emulsifiers, surfactants, thickening agents,coloring agents and combinations of two or more thereof.

Additionally, the combination of the invention can be administered inthe form of transdermal patches or iontophoresis devices. In oneembodiment, the combination of the invention is administered as atransdermal patch, e.g., in the form of sustained-release transdermalpatch. Suitable transdermal patches are known in the art.

Several drug delivery systems are known and can be used to administerthe combinations of the invention, including, e.g., encapsulation inliposomes, microbubbles, emulsions, microparticles, microcapsules andsimilars. The required dosage can be administered as a single unit or ina sustained release form.

Sustainable-release forms and appropriate materials and methods fortheir preparation are described in, e.g., “Modified-Release DrugDelivery Technology”, Rathbone, M. J. Hadgraft, J. and Roberts, M. S.(eds.), Marcel Dekker, Inc., New York (2002), “Handbook ofPharmaceutical Controlled 5 Release Technology”, Wise, D. L. (ed.),Marcel Dekker, Inc. New York, (2000). In one embodiment of theinvention, the orally administrable form of a combination orpharmaceutical composition of the invention is in a sustained releaseform further comprises at least one coating or matrix. The coating orsustained release matrix include, without limitation, natural polymers,semisynthetic or synthetic water-insoluble, modified, waxes, fats, fattyalcohols, fatty acids, natural semisynthetic or synthetic plasticizers,or a combination of two or more of them. In one embodiment, a pump canbe used (see Langer, supra: Sefton, 1987, CRC Crit. Ref. Biomed. Eng.14:201). In another embodiment, polymeric materials can be used see,Medical Applications of Controlled Release, Langer and Wise (eds.),1974, CRC Pres., Boca Raton, Fla. Other controlled release systems arediscussed in the review by Langer, 1990, Science 249:1527-1533.

Enteric coatings can be applied using conventional processes known toexperts in the art, as described in, e.g., Johnson, J. L.,“Pharmaceutical tablet coating”, Coatings Technology Handbook (SecondEdition), Satas, D. and Tracton, A. A. (eds), Marcel Dekker, Inc. NewYork, (2001), Carstensen, T., “Coating Tablets in AdvancedPharmaceutical Solids”, Swarbrick, J. (ed.), Marcel Dekker, Inc. NewYork (2001), 455-468.

A pharmaceutical composition of the present disclosure can be deliveredsubcutaneously or intravenously with a standard needle and syringe. Inaddition, with respect to subcutaneous delivery, a pen delivery devicereadily has applications in delivering a pharmaceutical composition ofthe present invention. Such a pen delivery device can be reusable ordisposable. A reusable pen delivery device generally utilizes areplaceable cartridge that contains a pharmaceutical composition. Onceall of the pharmaceutical composition within the cartridge has beenadministered and the cartridge is empty, the empty cartridge can readilybe discarded and replaced with a new cartridge that contains thepharmaceutical composition. The pen delivery device can then be reused.In a disposable pen delivery device, there is no replaceable cartridge.Rather, the disposable pen delivery device comes prefilled with thepharmaceutical composition held in a reservoir within the device. Oncethe reservoir is emptied of the pharmaceutical composition, the entiredevice is discarded.

Numerous reusable pen and autoinjector delivery devices haveapplications in the subcutaneous delivery of a pharmaceuticalcomposition of the present disclosure. Examples include, but are notlimited to AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen(Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75/25™pen, HUMALOG™ pen, HUMALIN 70/30™ pen (Eli Lilly and Co., Indianapolis,Ind.), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark),NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (BectonDickinson, Franklin Lakes, N.J.), OPTIPEN™, OPTIPEN PRO™, OPTIPENSTARLET™, and OPTICLlK™ (Sanofi-Aventis, Frankfurt, Germany), to nameonly a few. Examples of disposable pen delivery devices havingapplications in subcutaneous delivery of a pharmaceutical composition ofthe present invention include, but are not limited to the SOLOSTAR™ pen(Sanofi-Aventis), the FLEXPEN™ (Novo Nordisk), and the KWIKPEN™ (EliLilly), the SURECLICK™ Autoinjector (Amgen, Thousand Oaks, Calif.), thePENLET™ (Haselmeier, Stuttgart, Germany), the EPIPEN (Dey, L.P.), andthe HUMIRA™ Pen (Abbott Labs, Abbott Park III.), to name only a few.

IV. Medical Uses

In another aspect, the invention relates to a combination or apharmaceutical composition according to the invention for use inmedicine.

In another aspect, the invention relates to a combination or apharmaceutical composition according to the invention for use in theprevention and/or treatment of cancer.

Alternatively, the invention relates to the use of a combination or apharmaceutical composition of the invention for the preparation of amedicament for the prevention and/or treatment of cancer.

Alternatively, the invention relates to a method for preventing and/ortreating cancer comprising administering a combination or apharmaceutical composition of the invention to a subject in needthereof.

In another aspect, the invention relates to a combination or apharmaceutical composition according to the invention for use in theprevention and/or treatment of a hematological malignancy.

Alternatively, the invention relates to the use of a combination or apharmaceutical composition of the invention for the preparation of amedicament for the prevention and/or treatment of a hematologicalmalignancy.

Alternatively, the invention relates to a method for preventing and/ortreating a hematological malignancy comprising administering acombination or a pharmaceutical composition of the invention to asubject in need thereof.

In another aspect, the invention relates to a combination or apharmaceutical composition according to the invention for use in theprevention and/or treatment of a solid tumor.

Alternatively, the invention relates to the use of a combination or apharmaceutical composition of the invention for the preparation of amedicament for the prevention and/or treatment of a solid tumor.

Alternatively, the invention relates to a method for preventing and/ortreating a solid tumor comprising administering a combination or apharmaceutical composition of the invention in a subject in needthereof.

As used herein the terms “treat, ” “treatment, ” or “treatment of”refers to reducing the potential for a certain disease or disorder,reducing the occurrence of a certain disease or disorder, and/or areduction in the severity of a certain disease or disorder, preferably,to an extent that the subject no longer suffers discomfort and/oraltered function due to it. For example, “treating” can refer to theability of a therapy (i.e. the HTR1 modulator, e.g. the HTR1 antagonistand the antineoplastic agent, e.g. an antimetabolite antineoplasticagent) when administered to a subject, to prevent a certain disease ordisorder from occurring and/or to cure or to alleviate a certain diseasesymptoms, signs, or causes. “Treating” also refers to mitigating ordecreasing at least one clinical symptom and/or inhibition or delay inthe progression of the condition and/or prevention or delay of the onsetof a disease or illness. Thus, the terms “treat,” “treating” or“treatment of” (or grammatically equivalent terms) refer to bothprophylactic and therapeutic treatment regimes. Particularly,“treatment”, as used herein, relates to the administration of acombination according to the invention or of a pharmaceuticalcomposition according to the invention to a subject suffering from ahematological malignancy or a solid tumor including the administrationin an initial or early stage of a disease, wherein the object is toprevent or slow down (lessen) an undesired physiological change ordisorder.

The present disclosure provides methods and compositions generallyproviding a therapeutic benefit or desired clinical results. Atherapeutic benefit is not necessarily a cure for a particular diseaseor disorder, but rather encompasses a result which most typicallyincludes alleviation of the disease or disorder or increased survival,elimination of the disease or disorder, reduction or alleviation of asymptom associated with the disease or disorder, prevention oralleviation of a secondary disease, disorder or condition resulting fromthe occurrence of a primary disease or disorder, diminishment of extentof disease, stabilized (i.e., not worsening) state of disease, delay orslowing of disease progression, amelioration or palliation of thedisease state, and remission (whether partial or total), whetherdetectable or undetectable and/or prevention of the disease or disorder.Treatment also means prolonging survival as compared to expectedsurvival if not receiving the treatment.

The term “prevention”, “preventing” or “prevent”, as used herein,relates to the administration of a combination according to theinvention or of a pharmaceutical composition according to the inventionto a subject who has not been diagnosed as possibly having ahaematological malignancy or a solid tumor at the time ofadministration, but who would normally be expected to develop saiddisease or be at increased risk for said disease. The prevention intendsto avoid the appearance of said disease. The prevention can be complete(e.g. the total absence of a disease). The prevention can also bepartial, such that for example the occurrence of a disease in a subjectis less than that which would have occurred without the administrationof the combination or composition of the present invention. Preventionalso refers to reduced susceptibility to a clinical condition.

The term “subject” or “individual” or “animal” or “patient” includes anysubject, particularly a mammalian subject, for whom diagnosis,prognosis, or therapy is desired. Mammalian subjects include humans,domestic animals, farm animals, and zoo or pet animals such as dogs,cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows, and so on.In a particular embodiment of the invention, the subject is a mammal. Ina more particular embodiment of the invention, the subject is a human,particularly a human of any race and sex.

In some embodiments, a subject is a naïve subject. A naïve subject is asubject that has not been administered a therapy, e.g. an antineoplasticagent.

In another embodiment, a subject has received therapy and/or one or moredoses of a therapeutic agent to treat the haematological malignancy.

The term “haematological malignancy” refers to a type of cancer thataffects blood, bone marrow, and lymph nodes, and includes lymphomas,myelomas and leukemias. Historically, scientists and physicians haveclassified these diseases by their locations in the body, the appearanceof affected cells under the microscope, and the natural progression ofthe diseases. In leukemia, the cancerous cells are discoveredcirculating in the blood and bone marrow, while in lymphoma, the cellstend to aggregate and form masses, or tumors, in lymphatic tissues.Myeloma is a tumor of the bone marrow, and involves a specific subset ofwhite blood cells that produce a distinctive protein.

Haematological malignancies can derive from either of the two majorblood cell lineages: myeloid and lymphoid lineages. The myeloid lineagenormally produces granulocytes, erythrocytes, thrombocytes, macrophagesand mast cells; the lymphoid cell lineage produces B, T, NK lymphocytesand plasma cells. Non-limitative, illustrative examples ofhaematological malignancies are Acute lymphoblastic leukaemia (ALL),Acute myelogenous leukaemia (AML), Chronic lymphocytic leukaemia (CLL),Chronic myelogenous leukaemia (CML), Acute monocytic leukaemia (AMoL),Hodgkin’s lymphomas, non-Hodgkin’s lymphomas and myelomas.

In a particular embodiment, the hematological malignancy is a leukaemia.In a more particular embodiment, the leukaemia is selected from thegroup consisting of acute myeloid leukaemia (AML), acute lymphoblasticleukaemia (ALL), chronic lymphocytic leukaemia (CLL), and chronicmyeloid leukaemia (CML).

“Leukaemia”, as used herein, refers to a type of cancer of the blood orbone marrow characterized by an abnormal increase of immature whiteblood cells called “blasts” and that originates in blood-forming tissue.All leukaemias start in the bone marrow where developing blood cells,usually developing white cells, undergo a malignant (cancerous) change.This means that they multiply in an uncontrolled way crowding the marrowand interfering with normal blood cell production. Increasing numbers ofabnormal cells, called blast cells or leukaemic blasts eventually spillout of the bone marrow and travel around the body in the blood stream.In some cases these abnormal cells accumulate in various organsincluding the lymph nodes, spleen, liver and central nervous system(brain and spinal cord). Leukaemia is a broad term covering a spectrumof diseases and they are broadly classified by how quickly the diseasedevelops, and by the type of blood cell involved. In turn, it is part ofthe even broader group of diseases affecting the blood, bone marrow, andlymphoid system, which are all known as haematological neoplasms. Thereare four major kinds of leukaemia: Acute lymphoblastic leukaemia, orALL; Acute myeloid leukaemia, or AML; Chronic lymphocytic leukaemia, orCLL; Chronic myelogenous leukaemia, or CML.

“Acute Myeloid Leukaemia (AML) or acute myelogenous leukaemia or acutenonlymphocytic leukaemia (ANLL)” is a cancer of the myeloid line ofblood cells, characterized by the rapid growth of abnormal white bloodcells (myeloblasts) that accumulate in the bone marrow and interferewith the production of normal blood cells. The symptoms of AML arecaused by replacement of normal bone marrow with leukaemic cells, whichcauses a drop in red blood cells, platelets and normal white bloodcells. The combination of a myeloperoxidase or Sudan black stain and anonspecific esterase stain on blood and blood marrow smears are helpfulin distinguishing AML from ALL.

“Acute lymphoblastic leukaemia (ALL) or acute lymphoid leukaemia” is anacute form of leukaemia, or cancer of the white blood cells,characterized by the overproduction of cancerous, immature white bloodcells-known as lymphoblasts.

“Chronic lymphocytic leukaemia (CLL) or B-cell chronic lymphocyticleukaemia (B-CLL)” is a type of cancer that causes the body to producelarge numbers of white blood cells (B cell lymphocytes).

“Chronic myelogenous leukaemia (CML)” also known as “chronic myeloidleukaemia” or “chronic granulocytic leukaemia (CGL)” is a type of cancerthat causes the body to produce large numbers of white blood cells(myelocytes). In CML a proliferation of mature granulocytes(neutrophils, eosinophils and basophils) and their precursors is found.It is associated with a characteristic 15 chromosomal translocationcalled the Philadelphia chromosome.

In a particular embodiment, the hematological malignancy is a lymphoma.

“Lymphoma”, as used herein relates to a cancer that develops in thelymphatic system from cells called lymphocytes. Lymphomas arise whendeveloping lymphocytes undergo a malignant change and multiply in anuncontrolled way. Increasing numbers of abnormal lymphocytes, calledlymphoma cells accumulate and form collections of cancer cells calledmalignant tumours in lymph nodes and other parts of the body. There aredozens of subtypes of lymphomas, which are broadly divided into two maingroups: Hodgkin lymphoma (also known as Hodgkin’s disease) andNon-Hodgkin lymphomas (NHL) that can be divided into B-cell and T-celltype. In a particular embodiment, the lymphoma is Hodgkin’s lymphoma(HL). In another particular embodiment, the lymphoma is Non-Hodgkinlymphoma (NHL).

In another particular embodiment, the hematological malignancy is amyeloma.

“Myeloma” also known as “multiple myeloma” as used herein, is a cancerof plasma cells. Plasma cells are mature B-lymphocytes that livepredominantly in the bone marrow and normally produce antibodies. Inmyeloma, plasma cells undergo a malignant (cancerous) change andmultiply in an uncontrolled way producing large number of abnormalplasma cells, called myeloma cells and causing problems in differentparts of the body. There are several subtypes of myeloma. In each caseof myeloma, only one type of immunoglobulin is overproduced, but thisvaries from patient to patient. Types of myeloma are for exampleMonoclonal Gammopathy of Undetermined Significance (MGUS), AsymptomaticMyeloma, Smoldering Multiple Myeloma (SMM), Symptomatic or ActiveMyeloma, Light Chain Myeloma, Nonsecretory Myeloma and Plasmacytoma. Ina particular embodiment, the myeloma is selected from the groupconsisting of Monoclonal Gammopathy of Undetermined Significance (MGUS),Asymptomatic Myeloma, Smoldering Multiple Myeloma (SMM), Symptomatic orActive Myeloma, Light Chain Myeloma, Nonsecretory Myeloma andPlasmacytoma.

In a particular embodiment, the hematological malignancy is a myeloidneoplasm.

“Myeloid neoplasm”, as used herein, relates to clonal diseases ofhematopoietic stem or progenitor cells. The term “myeloid” includes allcells belonging to the granulocyte (i.e., neutrophil, eosinophil,basophil), monocyte/macrophage, erythroid, megakaryocyte, and mast celllineages. Such neoplasm can be present in the bone marrow and peripheralblood. They result from genetic and epigenetic alterations that perturbkey processes such as self-renewal, proliferation and impaireddifferentiation. Based on the morphology, cytochemistry,immunophenotype, genetics, and clinical features of myeloid disorders,the World Health Organization (WHO) categorizes myeloid neoplasms intofive primary types: (1) acute myeloid leukemia; (2) myelodysplasticsyndromes (MDS); (3) myeloproliferative neoplasms (MPN); (4)myelodysplastic and myeloproliferative (MDS/MPN) neoplasms; and (5)myeloid neoplasms associated with eosinophilia and abnormalities ofgrowth factor receptors derived from platelets or fibroblasts. In aparticular embodiment, the myeloid neoplasm is selected from the groupconsisting of acute myeloid leukemia (AML), myelodysplastic syndrome(MDS), myeloproliferative neoplasm (MPN), myelodysplastic andmyeloproliferative (MDS/MPN) neoplasm, and myeloid neoplasm associatedwith eosinophilia and abnormalities of growth factor receptors derivedfrom platelets or fibroblasts.

In a particular embodiment, the myeloid neoplasm is acute myeloidleukaemia.

In another particular embodiment, the myeloid neoplasm is amyelodysplastic syndrome.

“Myelodysplastic syndrome” (MDS), as used herein, refers to aheterogeneous group of closely related clonal hematopoietic disorderscommonly found in the aging population. All are characterized by one ormore peripheral blood cytopenias. Bone marrow is usually hypercellular,but rarely, a hypocellular marrow mimicking aplastic anemia can be seen.Bone marrow cells display aberrant morphology and maturation(dysmyelopoiesis), resulting in ineffective blood cell production. Thereare several different types of MDS and the disease can vary in itsseverity and the degree to which normal blood cell production isaffected. About 30% of people with MDS will progress to a form of cancercalled acute myeloid leukaemia (AML). It is sometimes referred to as apre-leukaemic disorder.

In another particular embodiment, the myeloid neoplasm is amyeloproliferative neoplasm.

“Myeloproliferative neoplasm (MPN)”, as used herein relates to clonalhematopoietic stem cell disorders characterized by proliferation of oneor more of the myeloid lineages. Each of these disorders involvesdysregulation at the multipotent hematopoietic stem cell (CD34) andclonal myeloproliferation and the absence of dyserythropoiesis,dysgranulopoiesis and monocytosis.

In another particular embodiment, the myeloid neoplasm is amyelodysplastic and myeloproliferative neoplasm.

“Myelodysplastic and myeloproliferative neoplasms (MDS/MPN)”, as usedherein, relates to clonal myeloid disorders that possess both dysplasticand proliferative features but are not properly classified as eithermyelodysplastic syndromes (MDS) or chronic myeloproliferative disorders(CMPD). The MDS/MPN category includes myeloid neoplasms with clinical,laboratory, and morphologic features that overlap MDS and MPN.

In another particular embodiment, the myeloid neoplasm is a myeloidneoplasm associated with eosinophilia and abnormalities of growth factorreceptors derived from platelets or fibroblasts.

“Myeloid neoplasms associated with eosinophilia and abnormalities ofgrowth factor receptors derived from platelets or fibroblasts”, as usedherein, relates to malignancies that arise by forming abnormal fusiongenes that encode altered surface or cytoplasmic proteins that activatesignal transduction pathways. There are different subtypes, all of themcharacterized by eosinophilia, although the clinical presentation ofeach subtype varies.

In another particular embodiment, the hematological malignancy is alymphoid neoplasm.

“Lymphoid Neoplasm”, as used herein relates to a disease derive from theclonal expansion and proliferation of B- and T-lymphocytes. Theyencompass a heterogeneous group of lymphomas and leukemias includingB-cell, T-cell, and natural killer (NK)-cell disorders.

In another particular embodiment, the hematological malignancy is atprecancerous stage. The term “precancerous stage”, as used herein,refers to one hyperproliferative disorder or premalignancy conditionthat can develop into cancer.

“Solid tumor” or solid cancers are neoplasms (new growth of cells) orlesions (damage of anatomic structures or disturbance of physiologicalfunctions) formed by an abnormal growth of body tissue cells other thanblood, bone marrow or lymphatic cells. A solid tumor consists of anabnormal mass of cells which may stem from different tissue types suchas liver, colon, breast, or lung, and which initially grows in the organof its cellular origin. However, such cancers may spread to other organsthrough metastatic tumor growth in advanced stages of the disease.

Examples of solid tumors are carcinomas, sarcomas, germinomas andblastomas.

Carcinomas are cancers derived from epithelial cells and account for 80%to 90% of all cancer cases since epithelial tissues are most abundantlyfound in the body. This group includes many of the most common cancers,particularly in the aged, and include lung cancer, colorectal cancer,pancreatic cancer, larynx cancer, tongue cancer, prostate cancer, breastcancer, ovarian cancer, liver cancer, head and neck cancer, esophagealcancer, renal cancer, endometrial cancer, gall bladder cancer, bladdercancer and gastric cancer. Carcinomas are of two types: adenocarcinomaand squamous cell carcinoma.

Adenocarcinoma develops in an organ or gland and squamous cell carcinomaoriginates in squamous epithelium. Adenocarcinomas may affect mucusmembranes and are first seen as a thickened plaque-like white mucosa.These are rapidly spreading cancers.

Sarcomas are cancers arising from connective tissue including muscles,bones, cartilage and fat. Examples of sarcomas include osteosarcoma (ofthe bone), chondrosarcoma (of the cartilage), leiomyosarcoma (smoothmuscles), rhabdomyosarcoma (skeletal muscles), mesothelial sarcoma ormesothelioma (membranous lining of body cavities), fibrosarcoma (fibroustissue), angiosarcoma or hemangioendothelioma (blood vessels),liposarcoma (adipose or fatty tissue), glioma or astrocytoma (neurogenicconnective tissue found in the brain), myxosarcoma (primitive embryonicconnective tissue) and mesenchymous or mixed mesodermal tumor (mixedconnective tissue types).

Germinomas refer to germ cell tumors, derived from pluripotent cells,most often presenting in the testicle or the ovary (seminoma anddysgerminoma, respectively).

Blastomas are cancers derived from immature precursor cells or embryonictissue. Blastomas are more common in children than in older adults.Examples of blastomas include hepatoblastoma, medulloblastoma,nephroblastoma, pancreatoblastoma, pleruropulmonary blastoma,retinoblastoma and glioblastoma multiforme.

Cancers that can be treated or prevented by the medical uses of thepresent invention are solid tumors, e.g. lung cancer, colorectal cancer,pancreatic cancer, larynx cancer, tongue cancer, breast cancer, ovariancancer, prostate cancer, liver cancer, head and neck cancer, esophagealcancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma,sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, dysgerminoma, embryonal carcinoma,Wilms’ tumor, cervical cancer, testicular tumor, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma.

Effectiveness of the combination and pharmaceutical composition of theinvention can be determined by analyzing the haematological response(measure the numbers of white cells, red cells and platelets and thelevels of hemoglobin and hematocrit), cytogenetic response and/orserological tumor markers.

In a particular embodiment, the cancer or hematological malignancy to betreated with the combination of the invention or the pharmaceuticalcomposition of the invention is a cancer or a hematological malignancycharacterized by having cells which express HTR1.

In order to determine if the cancer cell or hematological malignant cellexpresses HTR1, the expression of said receptor can be determined usingany method known in the art, such as those described in WO 2015/197839based on detecting type 1 HTR mRNA or protein in a sample as a whole, incells of a sample and/or in the non-cellular fraction of a sample.

The expression “detecting the expression” refers to detecting thepresence of a cancer cell or haematological cell in a sample carrying atype 1 HTR on its surface or expressing type 1 HTR mRNA. Said detectioncan be qualitative or quantitative.

The combinations and pharmaceutical compositions for use in the medicaltreatments according to the invention should be administered in apharmaceutical or therapeutically effective amount.

The expression “pharmaceutical or therapeutically effective amount”, asused herein, is understood as an amount capable of providing atherapeutic effect, some improvement or benefit to a subject having ahematological malignancy or a solid tumor. Thus, a “pharmaceutical ortherapeutically effective” amount is an amount that provides somealleviation, mitigation, and/or decrease in at least one clinicalsymptom of a hematological malignancy or a solid tumor. Those skilled inthe art will appreciate that therapeutic effects need not be complete orcurative, as long as some benefit is provided to the subject. The termalso encompasses the amount capable of reducing or preventing thehematological malignancy or a solid tumor. The pharmaceutically ortherapeutically effective amount can be determined by the person skilledin the art by commonly used means. The amount of the combination of theinvention or the pharmaceutical compositions according to the inventionwill vary depending upon the subject and the particular mode ofadministration. Those skilled in the art will appreciate that dosagescan also be determined with guidance from Goodman and Goldman’s ThePharmacological Basis of Therapeutics, Ninth Edition (1996), AppendixII, pp. 1707-1711 and from Goodman and Goldman’s The PharmacologicalBasis of Therapeutics, Tenth Edition (2001), Appendix II, pp. 475-493.

The appropriate dosage of the active principle or principles within thecombination or pharmaceutical composition will depend on the type ofcancer to be treated, the severity and course of the disease, whetherthe composition is administered for preventive or therapeutic purposes,previous therapy, the patient’s clinical history and response to thecompounds, and the discretion of the attending physician. The amount ofthe combination of the invention or the pharmaceutical compositionsaccording to the invention is suitably administered to the patient atone time or over a series of treatments. Depending on the type andseverity of the disease, an appropriate dosage level will generally beabout 0.01 to 500 mg per kg patient body weight per day which can beadministered in single or multiple doses. Particularly, the dosage levelwill be about 0.1 to about 250 mg/kg per day; more particularly about0.5 to about 100 mg/kg per day. A suitable dosage level can be about0.01 to 250 mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1to 50 mg/kg per day. Within this range the dosage can be 0.05 to 0.5,0.5 to 5 or 5 to 50 mg/kg per day. For oral administration, thecompositions are particularly provided in the form of tablets containing1.0 to 1000 milligrams of the active ingredient, particularly at leastabout 1.0, at least about 5.0, at least about 10.0, at least about 15.0,at least about 20.0, at least about 25.0, at least about 50.0, at leastabout 75.0, at least about 100.0, at least about 150.0, at least about200.0, at least about 250.0, at least about 300.0, at least about 400.0,at least about at least about 500.0, at least about 600.0, at leastabout 750.0, at least about at least about 800.0, at least about 900.0,and at least about 1000.0 milligrams of the active ingredient for thesymptomatic adjustment of the dosage to the patient to be treated.

For intravenous administration, the combination or the pharmaceuticalcompositions according to the invention is particularly administered ata dose of about 0.1 to about 10 mg per patient per day, moreparticularly from 0.1 to 9 mg per patient per day, more particularlyfrom 0.1 to 8 mg per patient per day, more particularly from 0.1 to 7,more particularly from 0.1 to 6 mg per patient per day, moreparticularly from 0.1 to 5 mg per patient per day mg per patient perday, more particularly from 0.1 to 4 mg per patient per day, moreparticularly from 0.1 to 3 mg per patient per day, more particularlyfrom 0.1 to 2 mg per patient per day, more particularly from 0.1 to 1 mgper patient per day. Dosages from 0.1 up to about 100 mg per patient perday may be used, particularly from 0.1 to 90 mg per patient per day,more particularly from 0.1 to 80 mg per patient per day, moreparticularly from 0.1 to 70 mg per patient per day, more particularlyfrom 0.1 to 60 mg per patient per day, more particularly from 0.1 to 50mg per patient per day, more particularly from 0.1 to 40 mg per patientper day, more particularly from 0.1 to 30 mg per patient per day, moreparticularly from 0.1 to 20 mg per patient per day; particularly whenthe drug is administered to a secluded site and not into the bloodstream, such as into a body cavity or into a lumen of an organ.Substantially higher dosages are possible in topical administration.

In a particular embodiment, the combination or the pharmaceuticalcomposition of the invention comprises an antineoplastic agent, e.g. anantimetabolite antineoplastic agent, in an amount between about 1 and3000 mg/m². Particularly, when the antimetabolite antineoplastic agentis a folic acid analogue, its amount in the combination or in thepharmaceutical composition is up to 3000 mg/m²; when the antimetaboliteantineoplastic agent is a pyrimidine analogue, its amount in thecombination or in the pharmaceutical composition is between about 1000and 3000 mg/m² (with the exception of decitabine, azacitidine andfluorouracil, which are under these amounts); when the antimetaboliteantineoplastic agent is a purine analogue, its amount in the combinationor in the pharmaceutical composition is between about 5 and 200 mg/m².

In a particular embodiment, the combination or the pharmaceuticalcomposition of the invention comprises cytarabine in an amount betweenabout 1 and 3000 mg/m². In a particular embodiment, the combination orthe pharmaceutical composition of the invention comprises decitabine inan amount between about 0.1 and 20 mg/m² In a particular embodiment, thecombination or the pharmaceutical composition of the invention comprisesazacitidine in an amount between 0.5 and 75 mg/m². In a particularembodiment, the combination or the pharmaceutical composition of theinvention comprises the HTR1 antagonist in an amount between about 0.1and 100 mg/m².

In particular embodiments, the combination or pharmaceutical compositioncomprises at least one HTR1 antagonist and at least one antimetaboliteantineoplastic agent in a ratio comprised between 500:1 and 2:1. Inparticular embodiments, the combination or pharmaceutical compositioncomprises at least one HTR1 antagonist and cytarabine in a ratiocomprised between 40:1 and 2:1. Examples of amounts of the HTR1antagonist (HTR1 antag) and cytarabine (cyt) in the combination or thepharmaceutical composition can be 50 mg/m² HTR1 antag + 1.25 mg/m²cyt;50 mg/m² HTR1 antag + 25 mg/m² cyt; 100 mg/m² HTR1 antag + 2.5 mg/m²cyt; 100 mg/m² HTR1 antag + 50 mg/m² cyt.

In particular embodiments, the combination or pharmaceutical compositioncomprises at least one HTR1 antagonist and azacitidine in a ratiocomprised between 100:1 and 2:1. Examples of amounts of the HTR1antagonist (HTR1 antag) and azacitidine (aza) in the combination or thepharmaceutical composition can be 50 mg/m² HTR1 antag + 0.5 mg/m² aza;50 mg/m² HTR1 antag + 25 mg/m² aza; 100 mg/m2 HTR1 antag + 1 mg/m² aza;100 mg/m² HTR1 antag + 50 mg/m² aza.

In particular embodiments, the combination or pharmaceutical compositioncomprises at least one HTR1 antagonist and decitabine in a ratiocomprised between 500:1 and 50:1. Examples of amounts of the HTR1antagonist (HTR1 antag) and decitabine (deci) in the combination or thepharmaceutical composition can be 50 mg/m² HTR1 antag + 0.1 mg/m² deci;50 mg/m² HTR1 antag + 1 mg/m² deci; 100 mg/m² HTR1 antag + 0.2 mg/m²deci; 100 mg/m² HTR1 antag + 2 mg/m² deci; 50 mg/m² HTR1 antag + 15mg/m² deci; 100 mg/m² HTR1 antag + 15 mg/m² deci.

In some embodiments, the combination or the pharmaceutical compositionaccording to the invention can be administered at a fixed dose. In otherembodiments, it can be administered as a variable dose. In someembodiments, it can be administered as a single dose. In otherembodiments, it can be administered in multiple doses, e.g. two or moredoses administered daily, weekly, biweekly, or monthly, particularlyonce or twice per day.

V. Method for Monitoring the Effect of the Therapy

This disclosure also provides methods for detecting the effectiveness ofthe combinations and pharmaceutical compositions of the inventionprovided herein through determining the protein expression level or geneexpression level of HTR1 as biormarkers. The inventors have found thatpresence of expression of HTR1 correlates with a good response totreatment with the combinations and the pharmaceutical compositionsprovided herein.

Thus, in another aspect, the invention relates to an in vitro method formonitoring the effect of the therapy (“therapy” hereinafter referred tothe combinations or the pharmaceutical compositions of the invention,i.e. the HTR1 modulator, e.g. the HTR1 antagonist and the antineoplasticagent, e.g. an antimetabolite antineoplastic agent) in a subjectsuffering from a haematological malignancy or a solid tumor and beingtreated with said combination or pharmaceutical composition whichcomprises:

-   a) Determining the expression level of type 1 HTR in cells of a    sample from said subject selected from the group consisting of bone    marrow, blood and lymph nodes, and-   b) Comparing said level with the expression level of type 1 HTR in    cells of a sample from said subject at an earlier point of time,    wherein a decrease of the expression level of type 1 HTR with    respect to the level determined in a sample from said subject at an    earlier point of time is indicative that the therapy is being    effective or wherein an increase of the expression level of type 1    HTR with respect to the level determined in a sample from said    subject at an earlier point of time is indicative that the therapy    is being ineffective.

The present disclosure also provides a method of treating a patienthaving a hematological malignancy or a solid tumor comprisingadministering the therapy if HTR1 expression is detected in a sampletaken from the patient, wherein said administration is effective toprevent and/or treat the hematological malignancy or the solid tumor inthe subject.

Also provided is a method of treating a patient having a hematologicalmalignancy or a solid tumor comprising (a) submitting a sample takenfrom the patient for measurement of the level of expression of HTR1, and(b) administering the therapy to the subject if HTR1 expression isdetected in the sample, wherein said administration is effective toprevent and/or treat the hematological malignancy or the solid tumor inthe subject.

In addition, the present disclosure provides a method of treating apatient having a hematological malignancy or a solid tumor comprising(a) measuring the level of expression of HTR1 in a sample taken from thepatient, (b) determining whether the patient’s level of expression ofHTR1 is above a predetermined HTR1 expression level of a one or morecontrol samples, and (c) advising a healthcare provider to administerthe therapy to the subject, wherein said administration is effective toprevent and/or treat the hematological malignancy or the solid tumor inthe subject.

Also provided is a method of determining whether to treat a patientdiagnosed with a hematological malignancy or a solid tumor comprising(a) measuring, or instructing a clinical laboratory to measure the levelof expression of HTR1 in a sample taken from the patient; and (b)treating, or instructing a healthcare provider to treat, the patient byadministering the therapy if HTR1 expression is detected in the sample,wherein said administration is effective to prevent and/or treat thehematological malignancy or the solid tumor in the subject.

The disclosure provides also a method of selecting a patient diagnosedwith a hematological malignancy or a solid tumor as a candidate fortreatment with the therapy comprising (a) measuring, or instructing aclinical laboratory to measure the level of expression of HTR1 in asample taken from the patient; and (b) treating, or instructing ahealthcare provider to treat the patient by administering the therapy ifHTR1 expression is detected in the sample, wherein said administrationis effective to prevent and/or treat the hematological malignancy or thesolid tumor in the subject.

In a preferred embodiment, the haematological malignancy is leukaemia,and more preferably acute myeloid leukaemia.

Methods for determining the expression level of type 1 5-HTR aredescribed in detail in WO2015/197839, methods which are incorporated byreference herein.

In a preferred embodiment, the in vitro method for monitoring the effectof the therapy in a subject suffering from a haematological malignancyor a solid tumor comprises determining the level of type 1 HTR. In amore preferred embodiment, the type 1 HTR is selected from the groupconsisting of HTR-1A and HTR-1B.

In a preferred embodiment, the method comprises determining theexpression level of HTR-1A and HTR-1B.

In another preferred embodiment, the blood sample is peripheral blood.

In another preferred embodiment, the in vitro method for monitoring theeffect of the therapy in a subject suffering from a haematologicalmalignancy or a solid tumor comprises determining the expression levelof type 1 HTR by measuring the level of mRNA encoded by the type 1 HTRgenes, or of variants thereof. In another preferred embodiment, theexpression level of type 1 HTR is determined by measuring the level oftype 1 HTR proteins, or of variants thereof.

In a more preferred embodiment, the mRNA expression level is determinedby PCR. In another preferred embodiment, the expression level ofproteins or of variants thereof is determined by Western blot orimmunocytochemistry. In a more preferred embodiment, the expressionlevel of type 1 HTR is determined by semi-quantitative PCR.

VI. Articles of Manufacture and Kits

The disclosure also provides articles of manufacture comprising any oneof the combinations or the pharmaceutical compositions disclosed herein,in one or more containers. In some embodiments, the article ofmanufacture comprises, e.g., a brochure, printed instructions, a label,or package insert directing the user (e.g., a distributor or the finaluser) to combine and/or use the compositions of the article ofmanufacture for the prevention and/or treatment of a hematologicalmalignancy or a solid tumor.

In some embodiments, the article of manufacture comprises, e.g.,bottle(s), vial(s), cartridge(s), box(es), syringe(s), injector(s), orany combination thereof. In some embodiments, the label refers to use oradministration of the combinations or the pharmaceutical compositions inthe article of manufacture according to the methods disclosed herein. Insome aspects, the label suggests, e.g., a regimen for use, a regimen fortreating, preventing, or ameliorating a hematological malignancy or asolid tumor.

This disclosure also provides kits for detecting the effectiveness ofthe combinations and pharmaceutical compositions provided herein (e.g.,to determine the protein expression level or gene expression level ofHTR)1, e.g., through an immunoassay method or nucleic acid detectionmethod. Such kits can comprise containers, each with one or more of thevarious reagents (e.g., in concentrated form) utilized in the method,including, e.g., one or more antibodies capable to specifically bindingto at least one biomarker (e.g., HTR1), or nucleic acid probes capableof specifically hybridizing to cDNA or mRNA for at least one biomarker.One or more antibodies against at least one biomarker, e.g., captureantibodies, or oligonucleotide probes can be provided already attachedto a solid support. One or more antibodies against at least onebiomarker, e.g., detection antibodies, or oligonucleotide probes can beprovided already conjugated to a detectable label, e.g., biotin or aruthenium chelate.

The kit can also provide reagents and instrumentation to support thepractice of the assays provided herein. In certain aspects, a labeledsecondary antibody can be provided that binds to the detection antibody.A kit provided according to this disclosure can further comprisesuitable containers, plates, and any other reagents or materialsnecessary to practice the assays provided herein.

In some embodiments, a kit comprises one or more nucleic acid probes(e.g., oligonucleotides comprising naturally occurring and/or chemicallymodified nucleotide units) capable of hybridizing a subsequence of abiomarker (HTR1) with high stringency conditions. In some aspects, oneor more nucleic acid probes (e.g., oligonucleotides comprising naturallyoccurring and/or chemically modified nucleotide units) capable ofhybridizing a subsequence of a biomarker under high stringencyconditions are attached to a microarray chip.

A kit provided according to this disclosure can also comprise brochuresor instructions describing the process. Test kits can includeinstructions for carrying out one or more biomarker detection assays,e.g., immunoassays or nucleic acid detection assays. Instructionsincluded in the kits can be affixed to packaging material or can beincluded as a package insert. While the instructions are typicallywritten or printed materials they are not limited to such. Any mediumcapable of storing such instructions and communicating them to an enduser is contemplated. Such media include, but are not limited to,electronic storage media (e.g., magnetic discs, tapes, cartridges,chips), optical media (e.g., CD ROM), and the like. As used herein, theterm “instructions” can include the address of an internet site thatprovides the instructions.

The contents of all cited references (including literature references,patents, patent applications, and websites) that may be cited throughoutthis application are hereby expressly incorporated by reference in theirentirety for any purpose, as are the references cited therein.

All terms as used herein, unless otherwise stated, shall be understoodin their ordinary meaning as known in the art. Other more specificdefinitions for certain terms as used in the present application are asset forth below and are intended to apply uniformly throughout thedescription and claims unless an otherwise expressly set out definitionprovides a broader definition. Throughout the description and claims theword “comprise” and variations of the word, are not intended to excludeother technical features, additives, components, or steps. Furthermore,the word “comprise” encompasses the case of “consisting of”. Additionalobjects, advantages and features of the invention will become apparentto those skilled in the art upon examination of the description or maybe learned by practice of the invention. Furthermore, the presentinvention covers all possible combinations of particular and particularembodiments described herein.

In this specification and the appended claims, the singular forms “a”,“an” and “the” include plural referents unless the context clearlydictates otherwise.

The terms “a” (or “an”), as well as the terms “one or more,” and “atleast one” can be used interchangeably herein. Furthermore, “and/or”where used herein is to be taken as specific disclosure of each of thetwo specified features or components with or without the other. Thus,the term “and/or” as used in a phrase such as “A and/or B” herein isintended to include “A and B,” “A or B,” “A” (alone), and “B” (alone).Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C”is intended to encompass each of the following aspects: A, B, and C; A,B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B(alone); and C (alone). The term “about” as used in connection with anumerical value throughout the specification and the claims denotes aninterval of accuracy, familiar and acceptable to a person skilled in theart. In general, such interval of accuracy is ± 15 %. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as commonly understood by one of ordinary skill in the art towhich this disclosure is related. For example, the Concise Dictionary ofBiomedicine and Molecular Biology, Juo, Pei-Show, 2nd ed., 2002, CRCPress; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999,Academic Press; and the Oxford Dictionary Of Biochemistry And MolecularBiology, Revised, 2000, Oxford University Press, provide one of skillwith a general dictionary of many of the terms used in this disclosure.Units, prefixes, and symbols are denoted in their Système Internationalde Unites (SI) accepted form. Numeric ranges are inclusive of thenumbers defining the range. Unless otherwise indicated, amino acidsequences are written left to right in amino to carboxy orientation. Theheadings provided herein are not limitations of the various aspects oraspects of the disclosure, which can be had by reference to thespecification as a whole. Accordingly, the terms defined immediatelybelow are more fully defined by reference to the specification in itsentirety.

The invention will be described by way of the following examples whichare to be considered as merely illustrative and not limitative of thescope of the invention.

EXAMPLES

Synergy is determined herein by means of the Combination Index Model,described previously (Chou TC, Pharmacological Reviews 58: 621-681,2006). The Cl was measured using Compusyn open software. Cl values below1 indicate synergism. Cl values about 1 indicate additivism and Clvalues above 1 indicate antagonisms.

The maximum concentrations corresponding to the EC50 (that dose whichproduces 50% cell death) and two lower doses corresponding to ½ and ⅕ ofthe EC50 have been chosen for each compound.

Example 1. Synergistic Effect of Cytarabine and HTR1 Antagonists

In order to study the effects of antineoplastic agents (cytostatic drug)in combination with HTR antagonists type 1, AML cell lines (TPH-1 andMonoMac1) were treated with different concentrations of HTR1 antagonists(apomorphine and methiothepin) in the presence of a range ofconcentrations of antineoplastics (cytarabine, azacytidine anddecitabine). Cytarabine is an antineoplastic agent widely used in thetreatment of lymphomas and leukemias.

Cell viability was measured by flow cytometry (live cells according tothe correct FSC-SSC profile, 7-AAD negativity and Hoechst 33342positivity) 72 h after treatment (FIG. 1A). As shown in FIG. 1B, bothapomorphine and methiothepin act synergically with cytarabine accordingto the combination index model (Tang et al. Front Pharmacol 2015;6:181).The synergic effect was detected in a molar range from 40:1 to 2:1 (HTR1antagonist:cytarabine).

Example 2- Synergistic Effect of Azacitidine and HTR1 Antagonists

Next, the inventors examined the interaction between HTR1 antagonistsand azacitidine. Azacitidine is a antineoplastic drug used for myeloidneoplasias. AML cell lines (HL-60 and MonoMac-1) were treated withdifferent concentrations of HTR1 antagonists (apomorphine andmethiothepin) in the presence of a wide range of concentrations ofazacitidine. Cell viability was measured by flow cytometry (live cellsaccording to the correct FSC-SSC profile, 7-AAD negativity and Hoechst33342 positivity) 72 h after treatment. As shown in FIG. 2A and FIG. 2B,both HTR1 antagonists displayed a synergically anti-leukemic effect froma molar relationship of 100:1 to 2:1 (HTR1:azacitidine).

Example 3- Synergistic Effect of Decitabine and HTR1 Antagonists

Similarly, decitabine was also interrogated for its potential cytotoxicinteraction with HTR1 antagonists. Decitabine is a cytostatic drugindicated for myeloid neoplasias. AML cell lines (HL-60 and MonoMac-1)were treated with different concentrations of HTR1 antagonists(apomorphine and methiothepin) in the presence of a range ofconcentrations of decitabine. 72 h after treatment, cell viability wasmeasured by flow cytometry (live cells according to the correct FSC-SSCprofile, 7-AAD negativity and Hoechst 33342 positivity). As shown inFIGS. 3A and 3B, both HTR1 antagonists displayed a synergicallyanti-leukemic effect from a molar relationship of 500:1 to 50:1 (HTR1:decitabine).

Example 4- Synergistic Effect in Vivo

In order to verify that the synergic cytotoxic effect was alsodetectable in vivo, pharmacological-conditioned NSG mice weretransplanted intravenously with 1 × 10⁶ MonoMac-1 AML cells. One weekafter transplantation, mice were treated for 5 days with 5 mg/kgapomorphine and/or 30 mg/kg cytarabine, both of them intraperitoneally.Mice were culled and bones were harvested and analyzed for humanleukemia by flow cytometry. As shown in FIG. 4 , Ara-C and apomorphinealso showed a synergistic effect (EOBA>25%) in in vivo treatment ofAML-bearing mice.

Example 5-Expression of HTR1A and HTR1B Correlates With CytarabineResistance

The sensitivity to HTR1 antagonists was studied in Ara-C-resistant-AMLcell lines (Cornet-Masana et al. Oncotarget 2016;7(17):23239-50). BothHL-60 and KG-1 parental cell lines displayed equivalent EC50 values forHTR1 antagonists (apomorphine and methiothepin) to Ara-C-resistant HL-60and KG15 1 (FIG. 5 ).

Example 6-HTR1A and HTR1B Expression in Relation to Cytarabine Treatment

Next, the expression of HTR1A and HTR1B on the surface by flow cytometrywas measured. Ara-C-resistant AML cells expressed higher levels of HTR1Aand HTR1B compared to the parental cell line (FIGS. s 6A and 6B). Infact, an inverse correlation between HTR1A and HTR1B expression leveland sensitivity to Ara-C was found (FIG. 6C); thus, AML cell lines withthe highest receptor expression showed the highest resistance to Ara-C.

Example 7-HTR1 Antagonists Decrease Sp1 Expression

At a gene expression level, the expression of Sp1 mRNA was studied. Sp1has been implicated with chemotherapeutic resistance (Zhang et al. MolCancer 2015;14:56). HL-60 AML cells were treated for 72 h with 10 µMapomorphine, 10 µM methiothepin and 5 µM SB224289. Total RNA as isolatedand mRNA was retrotranscribed to cDNA. Semi-quantitative PCR wasperformed with specific TaqMan oligo pairs. As shown in FIG. 7 , HTR1antagonist treatment produced the downregulation of Sp1 gene expression.

Example 8- Synergistic Effect of Cladribine and HTR1 Antagonists

Similar to the examples 1-3, cladribine was also interrogated for itspotential cytotoxic interaction with HTR1 antagonists. Cladribine is acytostatic drug indicated for myeloid neoplasias. AML cell line(MonoMac-1) was treated with different concentrations of HTR1antagonists (apomorphine and methiothepin) in the presence of a range ofconcentrations of cladribine. 72 h after treatment, cell viability wasmeasured by flow cytometry (live cells according to the correct FSC-SSCprofile, 7-AAD negativity and Hoechst 33342 positivity, andvolumetrically counted). As shown in FIGS. 8A and 8B, both HTR1antagonists displayed a synergically anti-leukemic effect from a molarrelationship of 100:1 to 1000:1 (HTR1:cladribine).

Example 9- Synergistic Effect of Fludarabine and HTR1 Antagonists

The potential cytotoxic interaction of fludarabine with HTR1 antagonistswas also examined on the same experimental conditions of the example 8.AML cell line (MonoMac-1) was treated with different concentrations ofHTR1 antagonists (apomorphine and methiothepin) in the presence of arange of concentrations of fludarabine. 72 h after treatment, cellviability was measured by flow cytometry (live cells according to thecorrect FSC-SSC profile, 7-AAD negativity and Hoechst 33342 positivity,and volumetrically counted). As shown in FIGS. 9A and 9B, both HTR1antagonists displayed a synergically antileukemic effect from a molarrelationship of 5:1 to 50:1 (HTR1:fludarabine).

Example 10- Synergistic Effect of Methotrexate and HTR1 Antagonist

Finally, the interaction of the antineoplastic drug methotrexate withHTR1 antagonist was studied. AML cell line (MonoMac-1) was treated withdifferent concentrations of HTR1 antagonist (apomorphine) in thepresence of a range of concentrations of methotrexate. 72 h aftertreatment, cell viability was measured by flow cytometry (live cellsaccording to the correct FSC-SSC profile, 7-AAD negativity and Hoechst33342 positivity, and volumetrically counted). As shown in FIG. 10 ,HTR1 antagonist displayed a synergically anti-leukemic effect from amolar relationship of 400:1 to 4000:1 (HTR1:methotrexate), in low andhigh concentrations.

1-14. (canceled)
 15. A combination for the treatment of a hematologicalmalignancy comprising (i) a type 1A or type 1B HTR serotonin receptor(HTR1A or HTR1B) small molecule antagonist; and. (ii) an ATC/DDD L01Bantimetabolite, wherein (i) and (ii) are present in a synergistic ratio,and wherein the combination can effectively kill hematologicalmalignancy cells expressing HTR1A or HTR1B.
 16. The combination of claim15, wherein the ATC/DDD L01B antimetabolite is a folic acid analogueselected from the group consisting of methotrexate, raltitrexed,pemetrexed, pralatrexate, and a combination thereof.
 17. The combinationof claim 15, wherein the ATC/DDD L01B antimetabolite is a pyrimidineanalogue selected from the group consisting of cytarabine, fluorouracil,tegafur, carmofur, gemcitabine, capecitabine, azacitidine, decitabine,trifluridine, and a combination thereof.
 18. The combination of claim15, wherein the ATC/DDD L01B antimetabolite is a purine analogueselected from the group consisting of mercaptopurine, tioguanine,cladribine, fludarabine, clofarabine, nelarabine, and combinationthereof.
 19. A pharmaceutical composition comprising a combination ofclaim 15 and a pharmaceutically acceptable excipient.
 20. A method fortreating a hematological malignancy comprising administering acombination of claim 15 to a subject in need thereof.
 21. The method ofclaim 20, wherein the hematological malignancy is a myeloid neoplasm.22. The method of claim 21, wherein the myeloid neoplasm is acutemyeloid leukemia.
 23. The method of claim 20, wherein the hematologicalmalignancy is selected from the group consisting of a myelodysplasticsyndrome and a myeloproliferative syndrome.
 24. The method of claim 20,wherein the ATC/DDD L01B antimetabolite and the HTR1A or HTR1B smallmolecule antagonist are administered independently or at the same time.25. The method of claim 20, wherein the HTR1A or HTR1B small moleculeantagonist is in an amount between 0.1 mg/m² and 100 mg/m².
 26. A kitcomprising a combination according to claim 15 in one or morecontainers.
 27. A method of manufacturing a combination for thetreatment of a hematological malignancy comprising mixing (i) an HTR1Aor HTR1B small molecule antagonist, and (ii) a ATC/DDD L01Bantimetabolite, wherein (i) and (ii) are present in a synergistic ratio,and wherein the combination can effectively kill hematologicalmalignancy cells expressing HTR1A or HTR1B.
 28. The method of claim 27,wherein the ATC/DDD L01B antimetabolite is a folic acid analogueselected from the group consisting of methotrexate, raltitrexed,pemetrexed, pralatrexate, and a combination thereof.
 29. The method ofclaim 27, wherein the ATC/DDD L01B antimetabolite is a pyrimidineanalogue selected from the group consisting of cytarabine, fluorouracil,tegafur, carmofur, gemcitabine, capecitabine, azacitidine, decitabine,trifluridine, and a combination thereof.
 30. The method of claim 27,wherein the ATC/DDD L01B antimetabolite is a purine analogue selectedfrom the group consisting of mercaptopurine, tioguanine, cladribine,fludarabine, clofarabine, nelarabine, and a combination thereof.
 31. Adosage form comprising a combination of (i) an HTR1A or HTR1B smallmolecule antagonist, and (ii) an ATC/DDD L01B antimetabolite comprising(a) a folic acid analogue selected from the group consisting ofmethotrexate, raltitrexed, pemetrexed, pralatrexate, and a combinationthereof; (b) a pyrimidine analogue selected from the group consisting ofcytarabine, fluorouracil, tegafur, carmofur, gemcitabine, capecitabine,azacitidine, decitabine trifluridine, and a combination thereof; (c) apurine analogue selected from the group consisting of mercaptopurine,tioguanine, cladribine, fludarabine, clofarabine, nelarabine, and acombination thereof; or, (d) a combination thereof, wherein (i) and (ii)are present in a synergistic ratio, and wherein the combination of (i)and (ii) can effectively kill hematological malignancy cells expressingHTR1A or HTR1B.
 32. The dosage form of claim 31, wherein the HTR1A orHTR1B small molecule antagonist is in an amount between 0.1 mg/m2 and100 mg/m2.
 33. The dosage form of claim 31, wherein the dosage form issuitable for intravenous administration.
 34. The dosage form of claim33, wherein the intravenous administration is by infusion or by bolusinjection.